OB fusion protein compositions and methods

ABSTRACT

The present invention relates to Fc-OB fusion protein compositions, methods of preparation of such compositions and uses thereof. In particular, the present invention relates to a genetic or chemical fusion protein comprising the Fc immunoglobulin region, derivative or analog fused to the N-terminal portion of the OB protein, derivative or analog.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of application Ser. No.09/568,528, filed May 9, 2000 and now pending, which is a continuationof application Ser. No. 09/267,517, filed Mar. 12, 1999 and nowabandoned, which is a continuation of application Ser. No. 08/770,973,filed Dec. 20, 1996 and now abandoned. Application Ser. No. 09/568,528is also a continuation-in-part of Ser. No. 09/094,931 filed Jun. 15,1998, and now pending, which is a continuation of application Ser. No.09/056,719, filed Apr. 7, 1998 and now abandoned, which is acontinuation of application Ser. No. 08/561,732, filed Nov. 22, 1995 andnow abandoned. All of these applications are incorporated by referenceas if contained herein in their entirety.

FIELD OF THE INVENTION

[0002] The present invention relates to Fc-OB fusion proteincompositions and methods for preparation and use thereof.

BACKGROUND

[0003] Although the molecular basis for obesity is largely unknown, theidentification of the “OB gene” and protein encoded (“OB protein” or“leptin”) has shed some light on mechanisms the body uses to regulatebody fat deposition. See, PCT publication, WO 96/05309 (Dec. 22, 1996),Friedman et al.; Zhang et al., Nature 372: 425-432 (1994); see also, theCorrection at Nature 374: 479 (1995). The OB protein is active in vivoin both ob/ob mutant mice (mice obese due to a defect in the productionof the OB gene product) as well as in normal, wild type mice. Thebiological activity manifests itself in, among other things, weightloss. See generally, Barrinaga, “Obese” Protein Slims Mice, Science.269: 475-456 (1995). The OB protein, derivatives and use thereof asmodulators for the control of weight and adiposity of animals, includingmammals and humans, has been disclosed in greater detail in PCTpublication WO 96/05309 (12/22/96), hereby incorporated by reference,including figures.

[0004] The other biological effects of OB protein are not wellcharacterized. It is known, for instance, that in ob/ob mutant mice,administration of OB protein results in a decrease in serum insulinlevels, and serum glucose levels. It is also known that administrationof OB protein results in a decrease in body fat. This was observed inboth ob/ob mutant mice, as well as non-obese normal mice. Pelleymounteret al., Science 269: 540-543 (1995); Halaas et al., Science 269: 543-546(1995). See also, Campfield et al., Science 269: 546-549(1995)(Peripheral and central administration of microgram doses of OBprotein reduced food intake and body weight of ob/ob and diet-inducedobese mice but not in db/db obese mice.) In none of these reports havetoxicity's been observed, even at the highest doses.

[0005] Despite the promise of clinical application of the OB protein,the mode of action of the OB protein in vivo is not clearly elucidated.Information on the OB receptor, shows high affinity binding of the OBprotein detected in the rat hypothalamus, which indicates OB receptorlocation. Stephens et al., Nature 377: 530-532. The db/db mouse displaysthe identical phenotype as the ob/ob mouse, i.e., extreme obesity andType II diabetes; this phenotype is thought to be due to a defective OBreceptor, particularly since db/db mice fail to respond to OB proteinadministration. See Stephens et al., supra.

[0006] With the advances in recombinant DNA technologies, theavailability of recombinant proteins for therapeutic use has engenderedadvances in protein formulation and chemical modification. One goal ofsuch modification is protein protection and decreased degradation.Fusion proteins and chemical attachment may effectively block aproteolytic enzyme from physical contact with the protein backboneitself, and thus prevent degradation. Additional advantages include,under certain circumstances, increasing the stability, circulation time,and the biological activity of the therapeutic protein. A review articledescribing protein modification and fusion proteins is Francis, Focus onGrowth Factors 3:4-10 (May 1992) (published by Mediscript, MountviewCourt, Friern Barnet Lane, London N20, OLD, UK).

[0007] One such modification is the use of the Fc region ofimmunoglobulins. Antibodies comprise two functionally independent parts,a variable domain known as “Fab”, which binds antigen, and a constantdomain, known as “Fc” which provides the link to effector functions suchas complement or phagocytic cells. The Fc portion of an immunoglobulinhas a long plasma half-life, whereas the Fab is short-lived. Capon, etal., Nature 337: 525-531 (1989).

[0008] Therapeutic protein products have been constructed using the Fcdomain to provide longer half-life or to incorporate-functions such asFc receptor binding, protein A binding, complement fixation andplacental transfer which all reside in the Fc proteins ofimmunoglobulins. Id. For example, the Fc region of an IgG1 antibody hasbeen fused to the N-terminal end of CD30-L, a molecule which binds CD30receptors expressed on Hodgkin's Disease tumor cells, anaplasticlymphoma cells, T-cell leukemia cells and other malignant cell types.See, U.S. Pat. No. 5,480,981. IL-10, an anti-inflammatory andantirejection agent has been fused to murine Fcγ2α in order to increasethe cytokine's short circulating half-life. Zheng, X. et al., TheJournal of Immunology, 154: 5590-5600 (1995). Studies have alsoevaluated the use of tumor necrosis factor receptor linked with the Fcprotein of human IgG1 to treat patients with septic shock. Fisher, C. etal., N. Engl. J. Med., 334: 1697-1702 (1996); Van Zee, K. et al., TheJournal of Immunology, 156: 2221-2230 (1996). Fc has also been fusedwith CD4 receptor to produce a therapeutic protein for treatment ofAIDS. See, Capon et al., Nature, 337:525-531 (1989). In addition, theN-terminus of interleukin 2 has also been fused to the Fc portion ofIgG1 or IgG3 to overcome the short half life of interleukin 2 and itssystemic toxicity. See, Harvill et al., Immunotechnology, 1: 95-105(1995).

[0009] Due to the identification of the OB protein as a promisingtherapeutic protein, there exists a need to develop OB analogcompositions for clinical application in conjunction with or in place ofOB protein administration. Such development would include OB analogcompositions where protein formulations and chemical modificationsachieve decreased protein degradation, increased stability andcirculation time. The present invention provides such compositions.

SUMMARY OF THE INVENTION

[0010] The present invention relates to Fc-OB fusion proteincompositions, methods of preparation of such compositions and usesthereof. In particular, the present invention relates to a geneticfusion protein comprising the Fc region or analogs of immunoglobulinsfused to the N-terminal portion of the OB protein or analogs. The Fc-OBfusion protein is capable of dimerizing via the cysteine residues of theFc region. Unexpectedly, genetic fusion modification with Fc at theN-terminus of the OB protein demonstrates advantages in stability,clearance rate and decreased degradation which are not seen in OBprotein or with fusion of Fc to the C-terminus of the OB protein.Surprisingly and importantly, the N-terminus modification providesunexpected protein protection from degradation, increases circulationtime and stability, when compared to the OB protein or Fc modificationto the OB protein C-terminus. Such unexpected advantages from the Fcmodification to OB protein would be advantageous to OB proteinconsumers, in that these changes contribute to lower doses required orless frequent dosing. Thus, as described below in more detail, thepresent invention has a number of aspects relating to The geneticmodification of proteins via fusion of the Fc region to the OB protein(or analogs thereof), as well as, specific modifications, preparationsand methods of use thereof.

[0011] Accordingly, in one aspect, the present invention provides aFc-OB fusion protein wherein Fc is genetically fused to the N-terminusof the OB protein. (or analogs thereof). In addition, the Fc portion mayalso be linked to the N-terminus of the OB protein (or analogs thereof)via peptide or chemical linkers as known in the art. As noted above anddescribed in more detail below, the Fc-OB fusion protein has unexpectedprotections from degradation and increased circulation time andstability when compared to the OB protein or C-terminus OB-Fc fusionproteins. Additional aspects of the present invention, therefore,include not only Fc-OB fusion protein compositions, but also DNAsequences encoding such proteins, related vectors and host cellscontaining such vectors, both useful for producing fusion proteins ofthe present invention.

[0012] In a second aspect, the present invention provides for preparingthe Fc-OB fusion protein. Such methods include recombinant DNAtechniques for preparation of recombinant proteins. Furthermore, suchaspects include methods of fermentation and purification as well.

[0013] In another aspect, the present invention provides methods fortreating excess weight in an individual or animals, including modulationof and/or fat deposition by the administration of Fc-OB fusion proteins.Due to the Fc-OB fusion protein characteristics, methods arecontemplated which reduce the amount and/or frequency of dosage of OBprotein by using Fc-OB weight reducing agent.

[0014] In yet another aspect, the present invention provides fortherapies for the treatment of co-morbidities associated with excessfat, such as diabetes, dys- or hyperlipidemias, arterial sclerosis,arterial plaque, the reduction or prevention of gall stones formation,stoke, and also an increase in insulin sensitivity and/or an increase inlean tissue mass.

[0015] In another aspect, the present invention also provides forrelated pharmaceutical compositions of the Fc-OB proteins, analogs andderivatives thereof, for use in the above therapies.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016]FIG. 1 Recombinant murine metoB (double stranded) DNA (SEQ. ID.NOs.:1 and 2) and amino acid sequence (SEQ. ID. NO. 3).

[0017]FIG. 2 Recombinant human metOB analog (double stranded) DNA (SEQ.ID. NOs.: 4 and 5) and amino acid sequence (SEQ. ID. NO. 6).

[0018] FIGS. 3(A-C) Recombinant human metFc-OB (double stranded) DNA(SEQ. ID. NOs.: 7 and 8) and amino acid sequence (SEQ. ID. NO. 9).

[0019] FIGS. 4(A-C) Recombinant human metFc-OB variant (double stranded)DNA (SEQ. ID. NOs.: 10 and 11) and amino acid sequence (SEQ. ID. NO.12).

[0020] FIGS. 5(A-C) Recombinant human-metFc-OB variant (double stranded)DNA (SEQ. ID. NOs.: 13 and 14) and amino acid sequence (SEQ. ID. NO.15).

[0021] FIGS. 6(A-C) Recombinant human metFc-OB variant (double stranded)DNA (SEQ. ID. NOs.: 16 and 17) and amino acid sequence (SEQ. ID. NO.18).

DETAILED DESCRIPTION

[0022] The present invention relates to Fc-OB fusion proteincompositions, methods of preparation of such compositions and usesthereof. In particular, the present invention relates to the genetic orchemical fusion of the Fc region of immunoglobulins to the N-terminalportion of the OB protein. Unexpectedly, fusion of Fc at the N-terminusof the OB protein demonstrates advantages which are not seen in OBprotein or with fusion of Fc at the C-terminus of the OB protein.Surprisingly, the N-terminally modified Fc-OB protein providesunexpected protein protection from degradation, increased circulationtime and increased stability. Accordingly, the Fc-OB fusion protein, andanalogs or derivatives thereof, as well as, related methods of use andpreparation, are described in more detail below.

[0023] Compositions

[0024] The Fc sequence of the recombinant human Fc-OB sequence set forthin SEQ. ID. NO. 9 (See FIG. 3) may be selected from the humanimmunoglobulin IgG-1 heavy chain, see Ellison, J. W. et al., NucleicAcids Res. 10: 4071-4079 (1982), or any other Fc sequence known in theart (e.g. other IgG classes including but not limited to IgG-2, IgG-3and IgG-4, or other immunoglobulins). Variant, analogs or derivatives ofthe Fc portion may be constructed by, for example, making varioussubstitutions of residues or sequences.

[0025] Cysteine residues can be deleted or replaced with other aminoacids to prevent formation of disulfide crosslinks of the Fc sequences.In particular amino acid at position 5 of SEQ. ID. NO. 9 is a cysteineresidue. The recombinant Fc-OB sequence of SEQ. ID. NO. 9 is a 378 aminoacid Fc-OB protein (not counting the methionine residue). The firstamino acid sequence for the recombinant Fc-OB protein of FIG. 3 isreferred to as+1 with the methionine at the −1 position.

[0026] One may remove the cysteine residue at position 5 or substituteit with one or more amino acids. An alanine residue may be substitutedfor the cysteine residue at position 6 giving the variant amino acidsequence of FIG. 4 (SEQ. ID. NO. 12). The recombinant Fc-OB protein ofFIG. 4 is a 378 amino acid Fc-OB protein (not counting the methionineresidue). The first amino acid sequence for the recombinant Fc-OBprotein of FIG. 4 is referred to as +1 with the methionine at the −1position.

[0027] Likewise, the cysteine at position 5 of SEQ. ID. NO. 9 could besubstituted with a serine or other amino acid residue or deleted. Avariant or analog may also be prepared by deletion of amino acids atpositions 1, 2, 3, 4 and 5 as with the variant in SEQ. ID. NO. 15 (SeeFIG. 5). Substitutions at these positions can also be made and are within the scope of this invention. The recombinant Fc-OB protein of FIG. 5is a 373 amino acid Fc-OB protein (not counting the methionine residue).The first amino acid sequence for the recombinant Fc-OB protein of FIG.5 is referred to as +1 with the methionine at the −1 position.

[0028] Modifications may also be made to introduce four amino acidsubstitutions to ablate the Fc receptor binding site and the complement(C1q) binding site. These variant modifications from SEQ. ID. NO. 15would include leucine at position 15 substituted with glutamate,glutamate at position 98 substituted with alanine, and lysines atpositions 100 and 102 substituted with alanines (see FIG. 6 and SEQ. ID.NO. 18). The recombinant Fc-OB protein of FIG. 6 is a 373 amino acidFc-OB protein (not counting the methionine residue). The first aminoacid sequence for the recombinant Fc-OB protein of FIG. 6 is referred toas +1 with the methionine at the −1 position.

[0029] Likewise, one or more tyrosine residues can be replaced byphenyalanine residues as well. In addition, other variant amino acidinsertions, deletions and/or substitutions are also contemplated and arewithin the scope of the present invention. Furthermore, alterations maybe in the form of altered amino acids, such as peptidomimetics orD-amino acids. The Fc protein may be also linked to the OB proteins ofthe Fc-OB protein by “linker” moieties whether chemical or amino acidsof varying lengths. Such chemical linkers are well known in the art.Amino acid linker sequences can include but are not limited to: (a) ala,ala, ala; (b) ala, ala, ala, ala; (c) ala, ala, ala, ala, ala; (d) gly,gly; (e) gly, gly, gly; (f) gly, gly, gly, gly, gly; (g) gly, gly, gly,gly, gly, gly, gly; (h) gly-pro-gly; (i) gly, gly, pro, gly, gly; and(j) any combination of subparts (a) through (i).

[0030] The OB portion of the Fc-OB fusion protein may be selected fromthe recombinant murine set forth in SEQ. ID. NO. 3 (See FIG. 1), or therecombinant human protein as set forth in Zhang et al., Nature, supra,(herein incorporated by reference) or those lacking a glutaminyl residueat position 28. (See Zhang et al, Nature, supra, at page 428.) One mayalso use the recombinant human OB protein analog as set forth in SEQ.ID. NO. 6 (See FIG. 2), which contains: (1) an arginine in place oflysine at position 35; and (2) a leucine in place of isoleucine atposition 74. (A shorthand abbreviation for this analog is therecombinant human R->L³⁵, I->L⁷⁴). The amino acid sequences for therecombinant human and recombinant murine proteins or analogs with orwithout-the fused Fc portion at the N-terminus of the OB protein are setforth below with a methionyl residue at the −1 position; however, aswith any of the present OB proteins and analogs, the methionyl residuemay be absent.

[0031] The murine protein is substantially homologous to the humanprotein, particularly as a mature protein., and, further, particularlyat the N-terminus. One may prepare an analog of the recombinant humanprotein by altering (such as substituting amino acid residues), in therecombinant human sequence, the amino acids which diverge from themurine sequence. Because the recombinant human protein has biologicalactivity in mice, such an analog would likely be active in humans. Forexample, using a human protein having a lysine at residue 35 and anisoleucine at residue 74 according to the numbering of SEQ. ID. NO. 6,wherein the first amino acid is valine, and the amino acid at position146 is cysteine, one may substitute with another amino acid one or moreof the amino acids at positions 32, 35, 50, 64, 68, 71, 74, 77, 89, 97,100, 105, 106, 107, 108, 111, 118, 136, 138, 142, and 145. One mayselect the amino acid at the corresponding position of the murineprotein, (SEQ. ID. NO. 3), or another amino acid.

[0032] One may further prepare “consensus” molecules based on the rat OBprotein sequence. Murakami et al., Biochem. Biophys. Res. Comm. 209:944-952 (1995) herein incorporated by reference. Rat OB protein differsfrom human OB protein at the following positions (using the numbering ofSEQ. ID. NO. 6): 4, 32, 33, 35, 50, 68, 71, 74, 77, 78, 89, 97, 100,101, 102, 105, 106, 107, 108, 111, 118, 136, 138 and 145. One maysubstitute with another amino acid one or more of the amino acids atthese divergent positions. The positions in bold print are those inwhich the murine OB protein as well as the rat OB protein are divergentfrom the human OB protein, and thus, are particularly suitable foralteration. At one or more of a positions, one may substitute an aminoacid from the corresponding rat OB protein, or another amino acid.

[0033] The positions from both rat and murine-OB protein which divergefrom the mature human OB protein are: 4, 32, 33, 35, 50, 64, 68, 71, 74,77, 78, 89, 97, 100, 102, 105, 106, 107, 108, 111, 118, 136, 138, 142,and 145. An OB protein according to SEQ. ID. NO. 6 having one or more ofthe above amino acids replaced with another amino acid, such as theamino acid found in the corresponding rat or murine sequence, may alsobe effective.

[0034] In addition, the amino acids found in rhesus monkey OB proteinwhich diverge from the mature human OB protein are (with identitiesnoted in parentheses in one letter amino acid abbreviation): 8 (S), 35(R), 48(V), 53(Q), 60(I), 66(I), 67(N), 68((L), 89(L), 100(L), 108(E),112 (D), and 118 (L). Since the recombinant human OB protein is activein cynomolgus monkeys, a human OB protein according to SEQ. ID. NO. 6(with lysine at position 35 and isoleucine at position 74) having one ormore of the rhesus monkey divergent amino acids replaced with anotheramino acid, such as the amino acids in parentheses, may be effective. Itshould be noted that certain rhesus divergent amino acids are also thosefound in the above murine species (positions 35, 68, 89, 100 and 112).Thus, one may prepare a murine/rhesus/human consensus molecule having(using the numbering of SEQ. ID. NO. 6 having a lysine at position 35and an isoleucine at position 74) having one or more of the amino acidsat positions replaced by another amino acid: 4, 8, 32, 33, 35, 48, 50,53, 60, 64, 66, 67, 68, 71, 74, 77, 78, 89, 97, 100, 102, 105, 106, 107,108, 111, 112, 118, 136, 138, 142, and 145.

[0035] Other analogs may be prepared by deleting a part of the proteinamino acid sequence. For example, the mature protein lacks a leadersequence (−22 to −1). One may prepare the following truncated forms ofhuman OB protein molecules (using the numbering of SEQ. ID. NO. 6):

[0036] (a) amino acids 98-146

[0037] (b) amino acids 1-32

[0038] (c) amino acids 40-116

[0039] (d) amino acids 1-99 and (connected to) 112-146

[0040] (e) amino acids 1-99 and (connected to) 112-146 having one ormore of amino acids 100-111 placed between amino acids 99 and 112.

[0041] In addition, the truncated forms may also have altered one ormore of the amino acids which are divergent (in the rat, murine, orrhesus OB protein) from human OB protein. Furthermore, any alterationsmay be in the form of altered amino acids, such as peptidomimetics orD-amino acids.

[0042] Therefore, the present invention encompasses a Fc-OB fusionprotein wherein the OB protein is selected from:

[0043] (a) the amino acid sequence 1-146 as set forth in SEQ. ID. NO. 3(below) or SEQ. ID. NO. 6;

[0044] (b) the amino acid sequence 1-146 as set forth in SEQ. ID. NO. 6having a lysine residue at position 35 and an isoleucine residue atposition 74;

[0045] (c), the amino acid sequence of subpart (b) having a differentamino acid substituted in one or more of the following positions (usingthe numbering according to SEQ. ID. NO. 6 and retaining the samenumbering even in the absence of a glutaminyl residue at position 28):4, 32, 33, 35, 50, 64, 68, 71, 74, 77, 78, 89, 97, 100, 102, 105, 106,107, 108, 111, 118, 136, 138, 142, and 145;

[0046] (d) the amino acid sequence of subparts (a), (b) or (c)optionally lacking a glutaminyl residue at position 28;

[0047] (e) the amino acid sequence of subparts (a), (b), (c), or (d)having a methionyl residue at the N-terminus;

[0048] (f) a truncated OB protein analog selected from among: (using thenumbering of SEQ. ID. NO. 6):

[0049] (i) amino acids 98-146

[0050] (ii) amino acids 1-32

[0051] (iii) amino acids 40-116

[0052] (iv) amino acids 1-99 and 112-146

[0053] (v) amino acids 1-99 and 112-146 having one or more of aminoacids 100-111 placed between amino acids 99 and 112; and,

[0054] (vi) the truncated OB analog of subpart (i) having one or more ofamino acids 100, 102, 105, 106, 107, 108, 111, 118, 136, 138, 142, and145 substituted with another amino acid;

[0055] (vii) the truncated analog of subpart (ii) having one or more ofamino acids 4, 8 and 32 substituted with another amino acid;

[0056] (viii) the truncated analog of subpart (iii) having one or moreof amino acids 50, 53, 60, 64, 66, 67, 68, 71, 74, 77, 78, 89, 97, 100,102, 105, 106, 107, 108, 111 and 112 replaced with another amino acid;

[0057] (vix) the truncated analog of subpart (iv) having one or more ofamino acids 4, 8, 32, 33, 35, 48, 50, 53, 60, 64, 66, 67, 68, 71, 74,77, 78, 89, 97, 112, 118, 136, 138, 142, and 145 replaced with anotheramino acid; and

[0058] (x) the truncated analog of subpart (v) having one or more ofamino acids 4, 32, 33, 35, 50, 64, 68, 71, 74, 77, 78, 89, 97, 100, 102,105, 106, 107, 108, 111, 118, 136, 138, 142, and 145 replaced withanother amino acid;

[0059] (xi) the truncated analog of any of subparts (i)-(x) having anN-terminal methionyl residue; and

[0060] (g) the OB protein or analog derivative of any of subparts (a)through (f) comprised of a chemical moiety connected to the proteinmoiety;

[0061] (h) a derivative of subpart (g) wherein said chemical moiety is awater soluble polymer moiety;

[0062] (i) a derivative of subpart (h) wherein said water solublepolymer moiety is polyethylene glycol;

[0063] (j) a derivative of subpart (h) wherein said water solublepolymer moiety is a polyaminoacid moiety;

[0064] (k) a derivative of subpart (h) through (j) wherein said moietyis attached at solely the N-terminus of said protein moiety; and

[0065] (l) an OB protein, analog or derivative of any of subparts (a)through (k) in a pharmaceutically acceptable carrier.

[0066] Derivatives

[0067] The present Fc-OB fusion proteins (herein the term “protein” isused to include “peptide,” Fc, OB or analogs, such as those recitedinfra, unless otherwise indicated) are derivatized by the attachment ofone or more chemical moieties to the Fc-OB fusion protein moiety. Thesechemically modified derivatives may be further formulated forintraarterial, intraperitoneal, intramuscular subcutaneous, intravenous,oral, nasal, pulmonary, topical or other routes of administration asdiscussed below. Chemical modification of biologically active proteinshas been found to provide additional advantages under certaincircumstances, such as increasing the stability and circulation time ofthe therapeutic protein and decreasing immunogenicity. See, U.S. Pat.No. 4,179,337, Davis et al., issued Dec. 18, 1979. For a review, seeAbuchowski et al., in Enzymes as Drugs. (J. S. Holcerberg and J.Roberts, eds. pp. 367-383 (1981)); Francis et al., supra.

[0068] The chemical moieties suitable for such derivatization may beselected from among various water soluble polymers. The polymer selectedshould be water soluble so that the protein to which it is attached doesnot precipitate in an aqueous environment, such as a physiologicalenvironment. Preferably, for therapeutic use of the end-productpreparation, the polymer will be pharmaceutically acceptable. Oneskilled in the art will be able to select the desired polymer based onsuch considerations as whether the polymer/protein conjugate will beused therapeutically, and if so, the desired dosage, circulation time,resistance to proteolysis, and other considerations. For the presentproteins and peptides, the effectiveness of the derivatization may beascertained by administering the derivative, in the desired form (i.e.,by osmotic pump, or, more preferably, by injection or infusion, or,further formulated for oral, pulmonary or nasal delivery, for example),and observing biological effects as described herein.

[0069] The water soluble polymer may be selected from the groupconsisting of, for example, polyethylene glycol, copolymers of ethyleneglycol/propylene glycol, carboxymethylcellulose, dextran, polyvinylalcohol, polyvinyl pyrolidone, poly-1, 3-dioxolane, poly-1,3,6-trioxane,ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymersor random copolymers), and dextran or poly(n-vinylpyrolidone)polyethylene glycol, propylene glycol homopolymers,polypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyolsand polyvinyl alcohol. Polyethylene glycol propionaldenhyde may haveadvantages in manufacturing due to its stability in water. Also,succinate and styrene may also be used.

[0070] The OB or Fc proteins used to formulate the Fc-OB fusion protein,may be prepared by attaching polyaminoacids or branch point amino acidsto the Fc or OB protein (or analogs) moiety. For example, thepolyaminoacid may be an additional carrier protein which, like the Fcfused to the OB protein or OB analog of the present invention, serves toalso increase the circulation half life of the protein in addition tothe advantages achieved via the Fc-OB fusion protein above. For thepresent therapeutic or cosmetic purpose of the present invention, suchpolyaminoacids should be those which have or do not create neutralizingantigenic response, or other adverse responses. Such polyaminoacids maybe selected from the group consisting of serum album (such as humanserum albumin), an additional antibody or portion thereof (e.g. the Fcregion), or other polyaminoacids, e.g. lysines. As indicated below, thelocation of attachment of the polyaminoacid may be at the N-terminus ofthe Fc-OB protein moiety, or C-terminus, or other places in between, andalso may be connected by a chemical “linker” moiety to the Fc-OBprotein.

[0071] The polymer may be of any molecular weight, and may be branchedor unbranched. For polyethylene glycol, the preferred molecular weightis between about 2 kDa and about 100 kDa (the term “about” indicatingthat in preparations of polyethylene glycol, some molecules will weighmore, some less, than the stated molecular weight) for ease in handlingand manufacturing. Other sizes may be used, depending on the desiredtherapeutic profile (e.g., the duration of sustained release desired,the-effects, if any on biological activity, the ease in handling, thedegree or lack of antigenicity and other known effects of thepolyethylene glycol to a therapeutic protein or analog).

[0072] The number of polymer molecules so attached may vary, and oneskilled in the art will be able to ascertain the effect on function. Onemay mono-derivatize, or may provide for a di-, tri-, tetra- or somecombination of derivatization, with the same or different chemicalmoieties (e.g., polymers, such as different weights of polyethyleneglycols). The proportion of polymer molecules to protein (or peptide)molecules will vary, as will their concentrations in the reactionmixture. In general, the optimum ratio (in terms of efficiency ofreaction in that there is no excess unreacted protein or polymer) willbe determined by factors such as the desired degree of derivatization(e.g., mono, di-, tri-, etc.), the molecular weight of the polymerselected, whether the polymer is branched or unbranched, and thereaction conditions.

[0073] The chemical moieties should be attached to the protein withconsideration of effects an functional or antigenic domains of theprotein. There are a number of attachment methods available to thoseskilled in the art. E.g., EP 0 401 384 herein incorporated by reference(coupling PEG to G-CSF), see also Malik et al., Exp. Hematol. 20:1028-1035 (1992) (reporting pegylation of GM-CSF using tresyl chloride).For example, polyethylene glycol may be covalently bound through aminoacid residues via a reactive group, such as, a free amino or carboxylgroup. Reactive groups are those to which an activated polyethyleneglycol molecule may be bound. The amino acid residues having a freeamino group may include lysine residues and the N-terminal amino acidresidue. Those having a free carboxyl group may include aspartic acidresidues, glutamic acid residues, and the C-terminal amino acid residue.Sulfhydryl groups may also be used as a reactive group for attaching thepolyethylene glycol molecule(s). Preferred for therapeutic purposes isattachment at an amino group, such as attachment at the N-terminus orlysine group. Attachment at residues important for receptor bindingshould be avoided if receptor binding is desired.

[0074] One may specifically desire N-terminally chemically modifiedFc-OB fusion protein. Using polyethylene glycol as an illustration ofthe present compositions, one may select from a variety of polyethyleneglycol molecules (by molecular weight, branching, etc.), the proportionof polyethylene glycol molecules to protein (or peptide) molecules inthe reaction mix, the type of pegylation reaction to be performed, andthe method of obtaining the selected N-terminally pegylated protein. Themethod of obtaining the N-terminally pegylated preparation (i.e.,separating this moiety from other monopegylated moieties if necessary)may be by purification of the N-terminally pegylated material from apopulation of pegylated protein molecules. Selective N-terminal chemicalmodification may be accomplished by reductive alkylation which exploitsdifferential reactivity of different types of primary amino groups(lysine versus the N-terminal) available for derivatization in aparticular protein. Under the appropriate reaction conditions,substantially selective derivatization of the protein at the N-terminuswith a carbonyl group containing polymer is achieved. For example, onemay selectively N-terminally pegylate the protein by performing thereaction at a pH which allows one to take advantage of the pK_(a)differences between the ε-amino group of the lysine residues and that ofthe α-amino group of the N-terminal residue of the protein. By suchselective derivatization, attachment of a water soluble polymer to aprotein is controlled: the conjugation with the polymer takes placepredominantly at the N-terminus of the protein and no significantmodification of other reactive groups, such as the lysine side chainamino groups, occurs. Using reductive alkylation, the water solublepolymer may be of the type described above, and should have a singlereactive aldehyde for coupling to the protein. Polyethylene glycolpropionaldehyde, containing a single reactive aldehyde, may be used.

[0075] An N-terminally monopegylated derivative is preferred for ease inproduction of a therapeutic. N-terminal pegylation ensures a homogenousproduct as characterization of the product is simplified relative todi-, tri- or other multi-pegylated products. The use of the abovereductive alkylation process for preparation of an N-terminal product ispreferred for ease in commercial manufacturing.

[0076] Complexes

[0077] The Fc-OB fusion protein, analog or derivative thereof may beadministered complexed to a binding composition. Such bindingcomposition may have the effect of prolonging the circulation time evenfurther than that achieved with the Fc-OB fusion protein, analog orderivative. Such composition may be a protein (or synonymously,peptide). An example of a binding protein is OB protein receptor orportion thereof, such as a soluble portion thereof. Other bindingproteins may be ascertained by examining OB protein or Fc-OB protein inserum, or by empirically screening for the presence of binding. Bindingproteins used will typically not interfere with the ability of OBprotein, Fc-OB fusion proteins, or analogs or derivatives thereof, tobind to endogenous OB protein receptor and/or effect signaltransduction.

[0078] Pharmaceutical Compositions

[0079] The present invention also provides methods of usingpharmaceutical compositions of the Fc-OB fusion proteins andderivatives. Such pharmaceutical compositions may be for administrationfor injection, or for oral, pulmonary, nasal, transdermal or other formsof administration. In general, comprehended by the invention arepharmaceutical compositions comprising effective amounts of protein orderivative products of the invention together with pharmaceuticallyacceptable diluents, preservatives, solubilizers, emulsifiers, adjuvantsand/or carriers. Such compositions include diluents of various buffercontent (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength;additives such as detergents and solubilizing agents (e.g., Tween 80,Polysorbate 80), anti-oxidants (e.g., ascorbic acid, sodiummetabisulfite), preservatives (e.g., Thimersol, benzyl alcohol) andbulking substances (e.g., lactose, mannitol); incorporation of thematerial into particulate preparations of polymeric compounds such aspolylactic acid, polyglycolic acid, etc. or into liposomes. Hylauronicacid may also be used, and this may have the effect of promotingsustained duration in the circulation. Such compositions may influencethe physical state, stability, rate of in vivo release, and rate of invivo clearance of the present proteins and derivatives. See, e.g.,Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack PublishingCo., Easton, Pa. 18042) pages 1435-1712 which are herein incorporated byreference. The compositions may be prepared in liquid form, or may be indried powder, such as lyophilized form. Implantable sustained releaseformulations are also contemplated, as are transdermal formulations.

[0080] Contemplated for use herein are oral solid dosage forms, whichare described generally in Remington's Pharmaceutical Sciences, 18th Ed.1990 (Mack Publishing Co. Easton Pa. 18042) at Chapter 89, which isherein incorporated by reference. Solid dosage forms include tablets,capsules, pills, troches or lozenges, cachets or pellets. Also,liposomal or proteinoid encapsulation may be used to formulate thepresent compositions (as, for example, proteinoid microspheres reportedin U.S. Pat. No. 4,925,673). Liposomal encapsulation may be used and theliposomes may be derivatized with various polymers (e.g., U.S. Pat. No.5,013,556). A description of possible solid dosage forms for thetherapeutic is given by Marshall, K. In: Modern Pharmaceutics Edited byG. S. Banker and C. T. Rhodes Chapter 10, 1979, herein incorporated byreference. In general, the formulation will include the Fc-OB fusionprotein (or analog or derivative), and inert ingredients which allow forprotection against the stomach environment, and release of thebiologically active material in the intestine.

[0081] Also specifically contemplated are oral dosage forms of the abovederivatized proteins. Fc-OB fusion protein may be chemically modified sothat oral delivery of the derivative is efficacious. Generally, thechemical modification contemplated is the attachment of at least onemoiety to the protein (or peptide) molecule itself, where said moietypermits (a) inhibition of proteolysis; and (b) uptake into the bloodstream from the stomach or intestine. Also desired is the increase inoverall stability of the protein and increase in circulation time in thebody. Examples of such moieties include: Polyethylene glycol, copolymersof ethylene glycol and propylene glycol, carboxymethyl cellulose,dextran, polyvinyl alcohol, polyvinyl pyrrolidone and polyproline.Abuchowski and Davis, Soluble Polymer-Enzyme Adducts. In: “Enzymes asDrugs”, Hocenberg and Roberts, eds., Wiley-Interscience, New York, N.Y.,(1981), pp 367-383; Newmark, at al., J. Appl. Biochem. 4: 185-189(1982). Other polymers that could be used are poly-1,3-dioxolane andpoly-1,3,6-tioxocane. Preferred for pharmaceutical usage, as indicatedabove, are polyethylene glycol moieties.

[0082] For the Fc-OB fusion protein, analog or derivative, the locationof release may be the stomach, the small intestine (e.g., the duodenum,jejunum, or ileum), or the large intestine. One skilled in the art hasavailable formulations which will not dissolve in the stomach, yet willrelease the material in the duodenum or elsewhere in the intestine.Preferably, the release will avoid the deleterious effects of thestomach environment, either by protection of the Fc-OB fusion protein,analog or derivative, or by release of the biologically active materialbeyond the stomach environment, such as in the intestine.

[0083] To ensure full gastric resistance a coating impermeable to atleast pH 5.0 is essential. Examples of the more common inert ingredientsthat are used as enteric coatings are cellulose acetate trimellitate(CAT), hydroxypropylmethylcellulose phthalate (HPMCP), HPMCP 50, HPMCP55, polyvinyl acetate phthalate (PVAP), Eudragit L30D, Aquateric,cellulose acetate phthalate (CAP), Eudragit L, Eudragit S, and Shellac.These coatings may be used as mixed films.

[0084] A coating or mixture of coatings can also be used on tablets,which are not intended for protection against the stomach. This caninclude sugar coatings, or coatings which make the tablet easier toswallow. Capsules may consist of a hard shell (such as gelatin) fordelivery of dry therapeutic i.e. powder; for liquid forms, a softgelatin shell may be used. The shell material of cachets could be thickstarch or other edible paper. For pills, lozenges, molded tablets ortablet triturates, moist massing techniques can be used.

[0085] The therapeutic can be included in the formulation as finemultiparticulates in the form of granules or pellets of particle sizeabout 1 mm. The formulation of the material for capsule administrationcould also be as a powder, lightly compressed plugs or even as tablets.The therapeutic could be prepared by compression.

[0086] Colorants and flavoring agents may all be included. For example,the protein (or derivative) may be formulated (such as by liposome ormicrosphere encapsulation) and then further contained within an edibleproduct, such as a refrigerated beverage containing colorants andflavoring agents.

[0087] One may dilute or increase the volume of the therapeutic with aninert material. These diluents could include carbohydrates, especiallymannitol, α-lactose, anhydrous lactose, cellulose, sucrose, modifieddextrans and starch. Certain inorganic salts may be also be used asfillers including calcium triphosphate, magnesium carbonate and sodiumchloride. Some commercially available diluents are Fast-Flo, Emdex,STA-Rx 1500, Emcompress and Avicell.

[0088] Disintegrants may be included in the formulation of thetherapeutic into a solid dosage form. Materials used as disintegratesinclude but are not limited to starch including the commercialdisintegrant based on starch, Explotab. Sodium starch glycolate,Amberlite, sodium carboxymethylcellulose, ultramylopectin, sodiumalginate, gelatin, orange peel, acid carboxymethyl cellulose, naturalsponge and bentonite may all be used. Another form of the disintegrantsare the insoluble cationic exchange resins. Powdered gums may be used asdisintegrants and as binders and these can include powdered gums such asagar, Karaya or tragacanth. Alginic acid and its sodium salt are alsouseful as disintegrants.

[0089] Binders may be used to hold the therapeutic agent together toform a hard tablet and include materials from natural products such asacacia, tragacanth, starch and gelatin. Others include methyl cellulose(MC), ethyl cellulose (EC) and carboxymethyl cellulose (CMC). Polyvinylpyrrolidone (PVP) and hydroxypropylmethyl cellulose (HPMC) could both beused in alcoholic solutions to granulate the therapeutic.

[0090] An antifrictional agent may be included in the formulation of thetherapeutic to prevent sticking during the formulation process.Lubricants may be used as a layer between the therapeutic and the diewall, and these can include but are not limited to; stearic acidincluding its magnesium and calcium salts, polytetrafluoroethylene(PTFE), liquid paraffin, vegetable oils and waxes; Soluble lubricantsmay also be used such as sodium lauryl sulfate, magnesium laurylsulfate, polyethylene glycol of various molecular weights, Carbowax 4000and 6000.

[0091] Glidants that might improve the flow properties of the drugduring formulation and to aid rearrangement during compression might beadded. The glidants may include starch, talc, pyrogenic silica andhydrated silicoaluminate.

[0092] To aid dissolution of the therapeutic into the aqueousenvironment a surfactant might be added as a wetting agent. Surfactantsmay include anionic detergents such as sodium lauryl sulfate, dioctylsodium sulfosuccinate and dioctyl sodium sulfonate. Cationic detergentsmight be used and could include benzalkonium chloride or benzethomiumchloride. The list of potential nonionic detergents that could beincluded in the formulation as surfactants are lauromacrogol 400,polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fattyacid ester, methyl cellulose and carboxymethyl cellulose. Thesesurfactants could be present in the formulation of the protein orderivative either alone or as a mixture in different ratios.

[0093] Additives which potentially enhance uptake of the protein (orderivative) are for instance the fatty acids oleic acid, linoleic acidand linolenic acid.

[0094] Controlled release formulation may be desirable. The drug couldbe incorporated into an inert matrix which permits release by eitherdiffusion or leaching mechanisms e.g., gums. Slowly degeneratingmatrices may also be incorporated into the formulation, e.g., alginates,polysaccahrides. Another form of a controlled release of thistherapeutic is by a method based on the Oros therapeutic system (AlzaCorp.), i.e., the drug is enclosed in a semipermeable membrane whichallows water to enter and push drug out through a single small openingdue to osmotic effects. Some enteric coatings also have a delayedrelease effect.

[0095] Other coatings may be used for the formulation. These include avariety of sugars which could be applied in a coating pan. Thetherapeutic agent could also be given in a film coated tablet and thematerials used in this instance are divided into 2 groups. The first arethe nonenteric materials and include methyl cellulose, ethyl cellulose,hydroxyethyl cellulose, methylhydroxy-ethyl cellulose, hydroxypropylcellulose, hydroxypropyl-methyl cellulose, sodium carboxy-methylcellulose, providone and the polyethylene glycols. The second groupconsists of the enteric materials that are commonly esters of phthalicacid.

[0096] A mix of materials might be used to provide the optimum filmcoating. Film coating may be carried out in a pan coater or in afluidized bed or by compression coating.

[0097] Also contemplated herein is pulmonary delivery of the presentprotein (or derivatives thereof). The protein (or derivative) isdelivered to the lungs of a mammal while inhaling and traverses acrossthe lung epithelial lining to the blood stream. (Other reports of thisinclude Adjei et al., Pharmaceutical Research 7: 565-569 (1990); Adjeiet al., International Journal of Pharmaceutics 63: 135-144(1990)(leuprolide acetate); Braquet et al., Journal of CardiovascularPharmacology 13 (suppl. 5): s.143-146 (1989)(endothelin-1); Hubbard etal., Annals of Internal Medicine 3: 206-212 (1989) (α1-antitrypsin);Smith et al., J. Clin. Invest. 84: 1145-1146 (1989)(a-1-proteinase);Oswein et al., “Aerosolization of Proteins”, Proceedings of Symposium onRespiratory Drug Delivery II, Keystone, Colorado, March, 1990(recombinant human growth hormone); Debs et al., The Journal ofImmunology 140: 3482-3488 (1988)(interferon-γ and tumor necrosis factora) and Platz et al., U.S. Pat. No. 5,284,656 (granulocyte colonystimulating factor).

[0098] Contemplated for use in the practice of this invention are a widerange of mechanical devices designed for pulmonary delivery oftherapeutic products, including but not limited to nebulizers, metereddose inhalers, and powder inhalers, all of which are familiar to thoseskilled in the art.

[0099] Some specific examples of commercially available devices suitablefor the practice of this invention are the Ultravent nebulizer,manufactured by Mallinckrodt, Inc., St. Louis, Mo.; the Acorn IInebulizer, manufactured by Marquest Medical Products, Englewood, Colo.;the Ventolin metered dose inhaler, manufactured by Glaxo Inc., ResearchTriangle Park, N.C.; and the Spinhaler powder inhaler, manufactured byFisons Corp., Bedford, Mass.

[0100] All such devices require the use of formulations suitable for thedispensing of protein (or analog or derivative). Typically, eachformulation is specific to the type of device employed and may involvethe use of an appropriate propellant material, in addition to diluents,adjuvants and/or carriers useful in therapy.

[0101] The protein (or derivative) should most advantageously beprepared in particulate form with an average particle size of less than10 μm (or microns), most preferably 0.5 to 5 μm, for most effectivedelivery to the distal lung.

[0102] Carriers include carbohydrates such as trehalose, mannitol,xylitol, sucrose, lactose, and sorbitol. Other ingredients for use informulations may include DPPC, DOPE, DSPC and DOPC. Natural or syntheticsurfactants may be used. Polyethylene glycol may be used (even apartfrom its use in derivatizing the protein or analog). Dextrans, such ascyclodextran, may be used. Bile salts and other related enhancers may beused. Cellulose and cellulose derivatives may be used. Amino acids maybe used, such as use in a buffer formulation.

[0103] Also, the use of liposomes, microcapsules or microspheres,inclusion complexes, or other types of carriers is contemplated.

[0104] Formulations suitable for use with a nebulizer, either jet orultrasonic, will typically comprise Fc-OB protein, analogs orderivatives thereof, dissolved in water at a concentration of about 0.1to 25 mg of biologically active protein per mL of solution. Theformulation may also include a buffer and a simple sugar (e.g., forprotein stabilization and regulation of osmotic pressure). The nebulizerformulation may also contain a surfactant, to reduce or prevent surfaceinduced aggregation of the protein caused by atomization of the solutionin forming the aerosol.

[0105] Formulations for use with a metered-dose Inhaler device willgenerally comprise a finely divided powder containing the protein (orderivative) suspended in a propellant with the aid of a surfactant. Thepropellant may be any conventional material employed for this purpose,such as a chlorofluorocarbon, a hydrochlorofluorocarbon, ahydrofluorocarbon, or a hydrocarbon, including trichlorofluoromethane,dichlorodifluoromethane, dichlorotetrafluoroethanol, and1,1,1,2-tetrafluoroethane, or combinations thereof. Suitable surfactantsinclude sorbitan trioleate and soya lecithin. Oleic acid may also beuseful as a surfactant.

[0106] Formulations for dispensing from a powder inhaler device willcomprise a finely divided dry powder containing protein (or derivative)and may also include a bulking agent, such as lactose, sorbitol,sucrose, mannitol, trehalose, or xylitol in amounts which facilitatedispersal of the powder from the device, e.g., 50 to 90% by weight ofthe formulation.

[0107] Nasal delivery of the protein (or analog or derivative) is alsocontemplated. Nasal delivery allows the passage of the protein to theblood stream directly after administering the therapeutic product to thenose, without the necessity for deposition of the product in the lung.Formulations for nasal delivery include those with dextran, orcyclodextran. Delivery via transport across other mucus membranes isalso contemplated.

[0108] Dosage

[0109] One skilled in the art will be able to ascertain effectivedosages by administration and observing the desired therapeutic effect.Due to the N-terminus modification of the OB protein, the presentinvention provides unexpected protein protection from degradation, andincreases circulation time and stability, when compared to OB protein orC-terminus modification of the OB protein. One skilled in the art,therefore, will be able to ascertain from these changes that aneffective dosage may require lower doses or less frequent dosing.

[0110] Preferably, the formulation of the molecule will be such thatbetween about 0.10 μg/kg/day and 10 mg/kg/day will yield the desiredtherapeutic effect. The effective dosages may be determined usingdiagnostic tools over time. For example, a diagnostic for measuring theamount of OB protein or Fc-OB fusion protein in the blood (or plasma orserum) may first be used to determine endogenous levels of protein. Suchdiagnostic tools may be in the form of an antibody assay, such as anantibody sandwich assay. The amount of endogenous OB protein isquantified initially, and a, baseline is determined. The therapeuticdosages are determined as the quantification of endogenous and exogenousOB protein or Fc-OB fusion protein (that is, protein, analog orderivative found within the body, either self-produced or administered)is continued over the course of therapy. The dosages may therefore varyover the course of therapy, with a relatively high dosage being usedinitially, until therapeutic benefit is seen, and lower dosages used tomaintain the therapeutic benefits.

[0111] Ideally, in situations where solely reduction in blood lipidlevels is desired, where maintenance of reduction of blood lipid levelsis desired, or an increase in lean body mass is desired, the dosage willbe insufficient to result in weight loss. Thus, during an initial courseof therapy of an obese person, dosages may be administered wherebyweight loss and concomitant blood lipid level lowering or concomitantfat tissue decrease/lean mass increase is achieved. Once sufficientweight loss is achieved, a dosage sufficient to prevent re-gainingweight, yet sufficient to maintain desired blood lipid levels or leanmass increase (or, prevention of lean mass depletion) may beadministered. These dosages can be determined empirically, as theeffects of OB or Fc-OB protein are reversible, (e.g., Campfield et al.,Science 269: 546-549 (1995) at 547). Thus, if a dosage resulting inweight loss is observed when weight loss is not desired, one wouldadminister a lower dose in order to achieve the desired blood lipidlevels or increase in lean tissue mass, yet maintain the desired weight.

[0112] For increasing an individual's sensitivity to insulin, similardosage considerations may be taken into account. Lean mass increasewithout weight loss may be achieved sufficient to decrease the amount ofinsulin (or, potentially, amylin, thiazolidinediones, or other potentialdiabetes treating drugs) an individual would be administered for thetreatment of diabetes.

[0113] For increasing overall strength, there may be similar dosageconsiderations. Lean mass increase with concomitant increase in overallstrength may be achieved with doses insufficient to result in weightloss. Other benefits, such as an increase in red blood cells (andoxygenation in the blood) and a decrease in bone resorption orosteoporosis may also be achieved in the absence of weight loss.

[0114] Combinations

[0115] The present methods may be used in conjunction with othermedicaments, such as those useful for the treatment of diabetes (e.g.,insulin, possibly, thiazolidinediones, amylin, or antagonists thereof),cholesterol and blood pressure lowering medicaments (such as those whichreduce blood lipid levels or other cardiovascular medicaments), andactivity increasing medicaments (e.g., amphetamines). Appetitesuppressants may also be used (such as those affecting the levels ofserotonin or neuropeptide Y). Such administration may be simultaneous ormay be in seriatim.

[0116] In addition, the present methods may be used in conjunction withsurgical procedures, such as cosmetic surgeries designed to alter theoverall appearance of a body (e.g., liposuction or laser surgeriesdesigned to reduce body mass). The health benefits of cardiac surgeries,such as bypass surgeries or other surgeries designed to relieve adeleterious condition caused by blockage of blood vessels by fattydeposits, such as arterial plaque, may be increased with concomitant useof the present compositions and methods. Methods to eliminate gallstones, such as ultrasonic or laser methods, may also be used eitherprior to, during or after a course of the present therapeutic methods.Furthermore, the present methods may be used as an adjunct to surgeriesor therapies for broken bones, damaged muscle, or other therapies whichwould be improved by an increase in lean tissue mass.

[0117] The following examples are offered to more fully illustrate theinvention, but are not to be construed as limiting the scope thereof.

EXAMPLE 1 Use of Murine FC-OB Protein Via Subcutaneous Injection

[0118] This example demonstrates that injection subcutaneously of murineFc-OB protein results in weight loss in normal mice. Normal (non-obese)CD1 mice were administered murine Fc-OB protein via subcutaneousinjections over a 22 day time period. A dosage of 10 mg protein/kg bodyweight/day resulted in a 14% (+/−1.1%) loss from baseline weight by the22nd day of injections. A dosage of PBS resulted in a 3.9% (+/−3.3%)loss from baseline weight by the 22nd day of injections. The weight losswith the use of 10 mg protein/kg body weight/day of Fc-OB protein inobese CD1 mice resulted in a 10% (+/−4.3%) loss from baseline weight anda dosage of PBS resulted in a 8.7% (+/−1.3%) loss from baseline weight,both by the 22nd day of injections

[0119] Presented below are the percent (%) differences from baselineweight in CD1 mice (8 weeks old): TABLE 1 Weight Loss Upon SubcutaneousInjection Lean/Recombinant Obese/Recombinant Time Fc-OB Fusion Fc-OBFusion (days) Vehicle (PBS) Protein Protein 1-2  −.44 +/ 1.1  −3.6 +/−.41 −1.03 +/− 1.36 3-4 −1.07 +/− .13  −6.8 +/− 1.5  −2.7 +/− 1.1 5-6 −.13 +/− 1.1  −9.5 +/− 1.2  −4.9 +/− .95 7-8  −.92 +/− .29 −12.5 +/−1.6  −7.7 +/− 2.9  9-10    1.6 +/− 1.3 −12.6 +/− 1.9  −8.2 +/− 2.9 11-12−1.98 +/− 1 −13.6 +/− 1.96  −8.6 +/− 2.9 13-14  −5.2 +/− 1.3 −14.6 +/−1.7 −10.1 +/− 3.6 15-16  −8.6 +/− 0.1 −14.5 +/− 2  −9.4 +/− 2.2 17-18 −8.5 +/− .64 −16.1 +/− 1.8  −9.6 +/− 2.99 19-20  −4.1 +/− .99   −16 +/−1.5 −10.4 +/− 3.3 21-22  −3.9 +/− 3.3 −14.1 +/− 1.1   −10 +/− 4.3

[0120] As can be seen, at the end of a 22 day subcutaneous regime,animals receiving the FC-OB protein lost over 14.1% of their body weightin lean and 10% of body weight in obese, as compared to animals onlyreceiving the PBS vehicle and as compared to baseline.

[0121] Surprisingly, animals receiving Fc-OB protein up to 22 dayscontinued to loose weight up until 28 days, 4 days after the lastinjection. Normal (non-obese) CD1 mice administered 10 mg protein/kgbody weight/day of murine Fc-OB protein via subcutaneous injectionsstopped at day 22 resulted in a 21% loss from baseline weight at day 28as compared to 14% loss at day 22. Likewise, obese CD1 mice administered10 mg protein/kg body weight/day of murine Fc-OB protein stopped at day22 resulted in a 13% loss from baseline weight at day 2.8 compared to10% loss at day 22. At day 34 weight loss was maintained at 10% loss inobese mice where lean mice recovered to 5% loss. Controls in each systemfrom day 22 through day 34 averaged from 4% in obese mice and 7% gain inlean mice.

EXAMPLE 2 Use of Human FC-OB Protein Via Subcutaneous Injection in C57Mice

[0122] This example demonstrates that injection subcutaneously of humanFc-OB protein results in weight loss in normal mice. Normal (non-obese)C57 mice were administered human Fc-OB protein via subcutaneousinjections over a 7 day time period. A dosage of 10 mg protein/kg bodyweight/day resulted in a 12% (+/−1.3%) loss from baseline weight by the7th day of injections. A dosage of 1 mg protein/kg body weight/dayresulted in a 8.9% (+/−1.5%) loss from baseline weight by the 7th day ofinjections. The weight loss with the use of 10 mg protein/kg bodyweight/day of human OB protein in obese C57 mice resulted in a 1.1%(+/−0.99%) loss from baseline weight and a dosage of 1 mg protein/kgbody weight/day resulted in a 2.5% (+/−1.1%) loss from baseline weight,both by the 7th day of injections.

[0123] Results

[0124] Presented below are the percent (%) differences from baselineweight in C57 mice (8 weeks old): TABLE 2 Weight Loss Upon SubcutaneousInjection Recombinant Time Fc-OB Fusion Recombinant OB (days) Vehicle(PBS) Protein Protein 1-2 .258 +/− 1.3  −6.4 +/− 1.6 −2.1 +/− .91 3-4 2.2 +/− 1.1 −12.1 +/− 1.5 −.78 +/− .36 5-6  4.5 +/− 2 −11.5 +/− 1.5−1.7 +/− .6 7-8  7.0 +/− 2.1 −11.9 +/− 1.6  0.1 +/− 1.2  9-10  9.0 +/−1.9 −11.5 +/− 1.3  7.2 +/− 2.7 11-12   10 +/− 3.8   −9 +/− 1.4 10.9 +/−2.9 13-14 12.5 +/− 4.4  −9.5 +/− 1.6 12.3 +/− 6.4 15-16 11.1 +/− 1.0 −3.0 +/− 1.5 10.3 +/− 3.3 17-18 17.2 +/− 3.6    8.0 +/− 1.3 13.3 +/−3.4

[0125] As can be seen, at the end of a day 17 after a 7 day subcutaneousregime at 10 mg/kg/day, animals receiving the FC-OB protein recovered to8% of their body weight. Animals receiving dosages of 1 mg/kg/day aftera 7 day subcutaneous regime returned to 6.4% of body weight after 12days.

[0126] These studies also show that during recovery periods from day 7to day 22, after the last injection at day 7, body weight recovery isslower in the Fc-OB treated C57 mice that with the OB treated mice. Thissuggests that the Fc-OB protein is not cleared as quickly as OB proteinthereby causing the extended weight loss effect.

EXAMPLE 3 Dose Response of CF7 Mice Treated with Fc-OB Fusion Protein:

[0127] An additional study demonstrated that there was a dose responseto continuous administration of Fc-OB protein. In this study, obese CF7mice, weighing 35-40 g were administered recombinant human Fc-OB proteinusing methods similar to the above example. The results are set forth inTable 3, below, (with % body weight lost as compared to baseline,measured as above): TABLE 3 Dose Response With Continuous Administration% Reduction in Body Dose Time Weight 0.25 mg/kg/day Day 5  4  0.5mg/kg/day Day 5 12   1 mg/kg/day Day 5 16

[0128] As can be seen, increasing the dose from 0.25 mg/kg/day to 1mg/kg/day increased the weight lost from 4% to 16%. It is alsonoteworthy that at day 5, the 1 mg/kg/day dosage resulted in a 16%reduction in body weight. These studies also showed slow weight recoveryrates to 0% suggesting that the Fc-OB protein is not quickly clearedthereby causing the extended weight loss effect.

EXAMPLE 4 Pharmacokinetics of Recombinant Human Fc-OB in CD-1 Mice andDogs

[0129] This study demonstrated the pharmacokinetic properties ofrecombinant human met Fc-OB protein in CD-1 mice and dogs. Followingintravenous or subcutaneous dosing at 1 mg/kg/day, serum concentrationsof recombinant human met Fc-OB protein and human met OB protein weredetermined by an enzyme-linked immunosorbent assay (ELISA).

[0130] In both species, an increase in exposure, as quantified by higherpeak serum concentrations and larger areasunder-the-serum-concentration-curve (AUC), was observed when compared torecombinant met-human OB protein. Fc-OB has lower systemic clearancethan recombinant met-human OB protein. This is seen in the lowerclearance and longer half-life of Fc-OB over OB protein. The increase insize causes not only an increase in protein stability, but also adecrease in the efficiency of renal clearance. As a result, Fc-OB iscleared slower from the systemic circulation. The increases in peaktime, peak serum concentrations and AUC for Fc-OB protein are consistentwith lower clearance. Fc-OB protein will yield substantially highersystemic exposure when compared to OB protein. Results are shown inTable 4 below: TABLE 4 Pharmacokinetic Properties Species CD-1 Mice CD-1Mice Beagle Dogs Route of Administration Intravenous SubcutaneousSubcutaneous OB Fc-OB OB Fc-OB OB Fc-OB protein protein protein proteinprotein protein Dose Level 1 1 1 1 0.5 0.5 (mg/kg) Peak Time (h) 0.14 62.8 8 Peak Serum 1520 7550 300 1120 Concentration (ng/mL) AUC (ng ·h/mL) 1470 366000 1230 132000 2200 52500 Half-life (h) 0.491 21.4 0.3882.13 22.9 Clearance 681 2.73 (mL/h/kg)

EXAMPLE 5

[0131] This example demonstrates that in normal mice which are not obeseand do not have elevated blood lipid levels, administration of humanrecombinant Fc-OB protein results in a lowering of cholesterol, glucoseand triglyceride levels. In addition, this example demonstrates thatthese levels remain low over a three day recovery period.

[0132] Normal CD1 mice were administered recombinant human Fc-OB proteinvia subcutaneous injections. Blood samples were taken 24 hours after day23, the last day of injection. As discussed above, the animals lostweight at the dosages administered. As shown in Table 5, the mice hadsubstantial reduction of serum cholesterol, glucose and triglycerides ina dose-dependent fashion when compared to controls: TABLE 5 Dose GlucoseCholesterol Triglycerides PBS 232.6 +/− 15.1 67.8 +/− 3.6 52.6 +/− 3.7  1 mg/kg/day 225.8 +/− 29.1   54 +/− 5.6   43 +/− 8.7 1.0 mg/kg/day193.2 +/− 21.4 53.4 +/− 5.7   38 +/− 11   1 mg/kg every 2 days 242.0 +/−9.3 52.6 +/− 4.4 40.8 +/− 7.2  10 mg/kg every 2 days 197.4 +/− 27.9 51.4+/− 5.9 29.8 +/− 6.3   1 mg/kg every 3 days 244.8 +/− 19.5 60.8 +/− 7.3  54 +/− 7.1  10 mg/kg every 3 days   188 +/− 31.2 52.2 +/− 6.9 26.2 +/−10.7

[0133] These data demonstrate that the Fc-OB protein, or analogs orderivatives thereof, are effective blood lipid lowering agents.

EXAMPLE 6

[0134] A obese human patient is administered human Fc-OB protein, oranalog or derivative for the purpose of weight reduction. The obesepatient also has elevated levels of blood lipids, including elevatedlevels of cholesterol, above 200 mg/100 ml. The patient attains asatisfactory weight reduction over the course of Fc-OB therapy. Amaintenance dose of Fc-OB protein or analog or derivative isadministered to the non-obese patient to maintain lowered blood lipidlevels, including lowered cholesterol levels, below 200 mg/100 ml. Thedose administered is insufficient to result in further weight loss.Administration is chronic. Levels of circulating Fc-OB protein or analogor derivative may be monitored using a diagnostic kit, such as anantibody assay against the OB protein (or other antigenic source ifapplicable).

EXAMPLE 7

[0135] A non-obese human patient undergoes coronary bypass surgery orother invasive treatment to alleviate advanced stages arterial plaqueformation. After the surgery, the patient is administered a maintenancedose of Fc-OB protein or analog or derivative in order to prevent there-formation of arterial plaque. The dose administered is insufficientto result in weight loss. Administration is chronic. Levels ofcirculating Fc-OB protein or analog or derivative may be monitored usinga diagnostic kit, such as an antibody assay against the OB protein (orother antigenic source if applicable).

EXAMPLE 8

[0136] A non-obese human patient experiences hypertension due torestricted blood flow from clogged arteries. The patient is administereda dose of Fc-OB protein, or analog or derivative thereof sufficient toreduce arterial plaque resulting in clogged arteries. Thereafter, thepatient is monitored for further arterial plaque formation, andhypertension. If the condition re-appears, the patient isre-administered an effective amount of Fc-OB protein, analog orderivative sufficient to restore blood flow, yet insufficient to resultin weight loss. Levels of circulating Fc-OB protein or analog orderivative may be monitored using a diagnostic kit, such as an antibodyassay against the Fc-OB protein (or other antigenic source ifapplicable).

EXAMPLE 9

[0137] A human patient experiences gall stones. Either the gall stonesare not removed and the formation of additional gall stones is sought tobe avoided, or the gall stones are removed but the gall bladder remains(as, for example, using laser or ultrasonic surgery) and the formationof additional gall stones is sought to be avoided. The patient isadministered an effective amount of Fc-OB protein, analog or derivativethereof to result in prevention of accumulation of additional gallstones or re-accumulation of gall stones. Levels of circulating Fc-OBprotein or analog or derivative may be monitored using a diagnostic kit,such as an antibody assay against the Fc-OB protein (or other antigenicsource if applicable).

EXAMPLE 10

[0138] A diabetic human patient desires to use decreased dosages ofinsulin for treatment of diabetes. The patient is administered aneffective amount of Fc-OB protein, analog or derivative thereof toresult in an increase in lean tissue mass. The patient's sensitivity toinsulin increases, and the dosage of insulin necessary to alleviatesymptoms of diabetes is decreased, either in terms of a decrease in theunits of insulin needed, or in terms of a decrease in the number ofinjections of insulin needed per day. Levels of Circulating Fc-OBprotein or analog or derivative may be monitored using a diagnostic kit,such as an antibody assay against the OB protein (or other antigenicsource if applicable).

EXAMPLE 11

[0139] A non-obese human patient desires an increase in lean tissue massfor therapeutic purposes, such as recovery from illness which depletedlean tissue mass. The patient is administered an effective amount ofFc-OB protein, analog or derivative thereof to result in the desiredincrease in lean tissue mass. Increase in lean tissue mass is monitoredusing DEXA scanning. Levels of circulating Fc-OB protein or analog orderivative may be monitored using a diagnostic kit, such as an antibodyassay against the OB protein (or other antigenic source if applicable).

[0140] Materials and Methods

[0141] Animals. Wild type CD1 mice and (+/+) C57B16 mice were used forthe above examples. The age of the mice at the initial time point was 8weeks, and the animals were weight stabilized.

[0142] Feeding and Weight Measurement. Mice were given ground rodentchow (PMI Feeds, Inc.) in powdered food feeders (Allentown Caging andEquipment) which allowed a more accurate and sensitive measurement thanuse of regular block chow. Weight was measured at the same time each day(2:00 p.m.), for the desired period. Body weight on the day prior to theinjection was defined as baseline weight. The mice used weighed 18-22grams.

[0143] Housing. Mice were single-housed, and maintained under humaneconditions.

[0144] Administration of Protein or Vehicle. Protein (as describedbelow) or vehicle (phosphate buffered saline, pH 7.4) were administeredby subcutaneous injections or intravenously.

[0145] Controls. Control animals were those who were injected with thevehicle alone without either Fc-OB fusion protein or OB protein added tothe vehicle.

[0146] Protein. Sequence ID. Nos. 1, 2 and 3 set forth murinerecombinant OB DNA and protein (FIG. 1), and Sequence ID. Nos. 4, 5 and6 set forth an analog recombinant human OB DNA and protein (FIG. 2). Asnoted above recombinant human OB protein as in SEQ. ID. NO. 6 has alysine residue at position 35 and an isoleucine residue at position 74.Furthermore, the recombinant human protein set forth in Zhang et al.,Nature, supra, and PCT publication WO 96/05309 (Dec. 22, 1996) (bothincorporated by reference including figures), and the murine and humananalog recombinant proteins of FIGS. 1 and 2 are illustrative of the OBprotein which may be used in forming the Fc-OB fusion protein of thepresent methods of treatment and manufacture of a medicament. Other OBor Fc proteins or analogs or derivatives thereof may also be used toform the Fc-OB fusion protein.

[0147] Herein, the first amino acid of the amino acid sequence forrecombinant OB protein is referred to as +1, and is valine, and theamino acid at position −1 is methionine. The C-terminal amino acid isnumber 146 (cysteine) (see FIGS. 1 and 2). The first amino acid sequencefor recombinant human Fc-OB protein of FIG. 3 is referred to as+1, andis glutamate, and the amino acid at position −1 is methionine. TheC-terminal amino acid is number 378 (cysteine). The first amino acidsequence for the recombinant human Fc-OB protein variant of FIG. 4 isreferred to as +1, and is glutamate, and the amino acid at position −1is methionine. The C-terminal amino acid is number 378 (cysteine). Thefirst amino acid sequence for the recombinant human Fc-OB proteinvariant of FIG. 5 is referred to as +1, and is aspartic acid, and theamino acid at position −1 is methionine. The C-terminal amino acid isnumber 373 (cysteine). The first amino acid sequence for the recombinanthuman Fc-OB protein variant of FIG. 6 is referred to as +1, and isaspartic acid, and the amino acid at position −1 is methionine. TheC-terminal amino acid is number is 373 (cysteine).

[0148] Expression Vector and Host Strain.

[0149] The plasmid expression vector used is pAMG21 (ATCC accessionnumber 98113), which is a derivative of pCFM1656 (ATCC accession number69576) and contains appropriate restriction sites for insertion of genesdownstream from the lux PR promoter (see U.S. Pat. No. 5,169,318 for adescription of the lux expression system). The FC-OB DNA, describedbelow and shown in FIGS. 3-6, was created and ligated into theexpression vector pAMG21 linearized with restriction endonucleases NdeIand BamHI and transformed into the E. coli host strain, FM5. E. coli FM5cells were derived at Amgen Inc., Thousand Oaks, Calif. from E. coliK-12 strain (Bachmann, et al., Bacterial. Rev. 40: 116-167 (1976)) andcontain the integrated lambda phage repressor gene, cI₈₅₇ (Sussman etal., C. R. Acad. Sci. 254: 1517-1579 (1962)). Vector production, celltransformation, and colony selection were performed by standard methods,(e.g., Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2dEdition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.)Host cells were grown in LB media.

[0150] Fc-OB DNA Construction

[0151] The plasmid pFc-A3 (described below) served as the source ofsequence for human immunoglobulin IgG-1 heavy chain from amino acidnumber 99. Glu) to the natural carboxyl terminus. The human IgG-1sequence can be obtained from Genebank (P01857).

[0152] The human OB sequence is disclosed above as well as Zhang et al.,Nature, supra, and PCT publication WO 96/05309 both incorporated byreference including drawings. The OB DNA was ligated into the expressionvector pCFM1656 linearized with restriction endonucleases XbaI and BamHIusing standard cloning procedures., e.g., Sambrook, et al., MolecularCloning: A Laboratory Manual, 2d Edition, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. The plasmid pCFM1656 carrying the OB DNAsequence served as the source of sequence for the recombinant human OBgene.

[0153] The genetic fusing of these two sequences was carried out by themethod of PCR overlap extension (Ho, S. N., et al., Site DirectedMutagenesis By Overlap Extension Using The Polymerase Chain Reaction,Gene 77:51-59(1989)). The product of the PCR was cleaved withrestriction endonuclease NdeI to create a 5′-cohesive end and withrestriction endonuclease BamHI to create a 3′-cohesive terminus. Thevector, pAMG21, was similarly cleaved. A ligation was performed with thefusion fragment and the linearized vector. Ligated DNA was transformedby electroporation into the E. coli host strain., Clones surviving onkanamycin (50 μg/ml) selection agar plates were checked for expressionof Fc-OB-sized protein. Plasmid from individual clones was isolated andthe sequence of the gene coding region verified.

[0154] When additional modifications of the Fc-OB gene were desired, thePCR technique was used again to engineer the changes. Two sets ofchanges were performed at the N-terminus of the Fc portion of the fusionprotein (SEQ. ID. No. 9) to create the variants SEQ. ID. NOS. 12 and 15.Another variant was constructed to introduce four amino acidsubstitutions to ablate the Fc-receptor binding site (leucine atposition 15 substituted with glutamate), and the complement (C1q)binding site (glutamate at position 98 substituted with alanine, lysineat position 100 substituted with alanine, and lysine at position 102substituted with alanine (See, Xin Xiao Zheng et. al, J. Immunol. 154:5590-5600 (1995)). The template for this construct was Seq. ID. No. 15and the resulting variant was SEQ. ID. Nos. 18.

[0155] pFC-A3 Vector Construction

[0156] A plasmid, pFc-A3, containing the region encoding the Fc portionof human immunoglobulin IgG-1 heavy chain (See Ellison, J. W. et. al,Nucleic Acids Res. 10:4071-4079 (1982)), from the first amino acidGlu-99 of the hinge domain to the carboxyl terminus plus a 5′-NotIfusion site and 3′-SalI and XbaI sites, was made by PCR amplification ofthe human spleen cDNA library. PCR reactions were in a final volume of100 ml and employed 2 units of Vent DNA polymerase in 20 mM Tris-HCl (pH8.8), 10 mM KCl, 10 mM (NH₄)₂SO₄, 2 mM MgSO₄, 0.1% Triton X-100 with 400mM each dNTP and 1 ng of the cDNA library to be amplified together with1 uM of each primer. Reactions were initiated by denaturation at 95° C.for 2 min, followed by 30 cycles of 95° C. for 30 s, 55° C. for 30 s,and 73° C. for 2 min. The 5′-primer incorporated a NotI site immediately5′ to the first residue (Glu-99) of the hinge domain of IgG-1. The3′-primer incorporated SalI and XbaI sites. The 717 base pair PCRproduct was digested with NotI and SalI, the resulting DNA fragment wasisolated by electrophoresis through 1% agarose and purified and clonedinto NotI, SalI-digested pBluescript II KS vector (Stratagene). Theinsert in the resulting plasmid, pFc-A3, was sequenced to confirm thefidelity of the PCR reaction.

[0157] Methods for Production

[0158] The methods below for production have been used to producebiologically active recombinant methionyl murine or human analog OBprotein and Fc-OB fusion proteins. Similar methods may be used toprepare biologically active methionyl human OB protein.

[0159] Fermentation Process

[0160] A batch fermentation process was used. Media compositions are setforth below.

[0161] A portion of the media consisting of primarily nitrogen sourceswas sterilized (by raising temperature to 120-123° C. for 25-35 minutes)in the fermentation vessel. Upon cooling, carbon, magnesium, phosphate,and trace metal sources were added aseptically. An overnight culture ofthe above recombinant murine protein-producing bacteria of 500 mL (grownin LB broth) was added to the fermentor. When the culture opticaldensity (measured at 600 nm as an indicator for cell density) reached15-25 absorption units, an autoinducer solution (0.5 mg/mL homoserinelactone) was added (1 mL/L) to the culture to induce the recombinantgene expression. The fermentation process was allowed to continue foradditional 10 to 16 hours, followed by harvesting the broth bycentrifugation.

[0162] Media Composition:

[0163] Batch:

[0164] 34 g/L Yeast extract

[0165] 78 g/L Soy peptone

[0166] 0.9 g/L Potassium chloride

[0167] 5.0 g/L Hexaphos

[0168] 1.7 g/L Citric acid

[0169] 120 g/L Glycerol

[0170] 0.5 g/L MgSO₄.7H₂O

[0171] 0.2 mL/L Trace Metal Solution

[0172] 0.5 mL/L P2000 Antifoam

[0173] Trace Metal Solution:

[0174] Ferric Chloride (FeCl₃.6H₂O): 27 g/L

[0175] Zinc Chloride (ZnCl₂.4H₂O): 2 g/L

[0176] Cobalt Chloride (CoCl₂.6H₂O): 2 g/L

[0177] Sodium Molybdate (NaMoO₄.2H₂O): 2 g/L

[0178] Calcium Chloride (CaCl₂.2H₂O): 1 g/L

[0179] Cupric Sulfate (CuSO₄.5H₂O): 1.9 g/L

[0180] Boric Acid (H₃BO₃): 0.5 g/L

[0181] Manganese Chloride (MnCl₂.4H₂O): 1.6 g/L

[0182] Sodium Citrate dihydrate: 73.5 g/L

[0183] Purification Process for Human Fc-OB Fusion Protein

[0184] Purification for human Fc-OB fusion protein was accomplished bythe steps below (unless otherwise noted, the following steps wereperformed at 4° C.). Purification for murine and human OB protein isdisclosed in PCT publication WO 96/05309, supra, herein incorporated byreference.

[0185] 1. Cell paste. E. coli cell paste was suspended in 5 timesvolumes of distilled water. The cells in the water were further brokenby two passes through a microfluidizer. The broken cells werecentrifuged at 4.2k rpm for 1 hour in a Beckman JB-6 centrifuge with aJ5-4.2 rotor.

[0186] 2. Inclusion body wash. The supernatant from above was removedand the pellet was resuspended with five volumes of distilled water. Themixture was centrifuged as in step 1.

[0187] 3. Solubilization. The pellet was solubilized with 10 volumes of50 mM tris, pH 8.5, 8 M guanidine hydrochloride, 10 mM dithiothreitoland stirred for one hour at room temperature. The solution is made 40 mMcystamine dihydrochloride and stirred for one hour.

[0188] 4. The solution from step 3 is added to 20 to 30 volumes of thefollowing refold solution 50 mM tris, pH 8.5, 0.8 M arginine, 2 M urea,and 4 mM cysteine. The refold is stirred for 16 hours at 8° C.

[0189] 5. Buffer exchange. The solution from step 4 is concentrated anddiafiltered into 10 mM tris, pH 8.5.

[0190] 6. Acid precipitation. The solution from step 5 is adjusted to pH4.75 with 50% glacial acid and incubated for 30 minutes at roomtemperature. The solution is filtered.

[0191] 7. Cation exchange chromatography. The solution from step 6 isadjusted to pH 7.0 and loaded onto a CM Sepharose Fast Flow column at10° C. A twenty column volume gradient is done at 10 mM phosphate, pH7.0, 0 to 0.1 M NaCl.

[0192] 8. Anion exchange chromatography. The CM elution pool from step 7is diluted 5 fold with 5 mM tris, pH 7.5 and loaded onto a Q SepharoseFast Flow at 10′ C. A 20 column volume gradient is done at 10 mM tris,pH 7.5, 0 to 0.2M NaCl.

[0193] 9. Hydrophobic interaction chromatography. The Q sepharose poolis made 0.75M ammonium sulfate and loaded on a methyl Macroprephydrophobic interaction column at room temperature. A 20 column volumegradient is done at 10 mM phosphate, pH 7.0, 0.75M to 0M ammoniumsulfate.

[0194] 10. Buffer exchange. The pool from step 9 is concentrated asnecessary and dialyzed against PBS buffer.

[0195] While the present invention has been described in terms ofpreferred embodiments, it is understood that variations andmodifications will occur to those skilled in the art. Therefore, it isintended that the appended claims cover all such equivalent variationswhich come within the scope of the invention as claimed.

1 16 1 499 DNA Phaseolus lunatus 1 gcgaagatct gagagaatgg cgaggtctagcagcaaggat gcacaagacc ttttccgagc 60 tctttggtct gcttatgccg caacccccacaaatctcaag atcattgacc tctacgtcat 120 gttcgctgtt ttcaccgctc tcatccaggtagtttacatg gctttggtgg gatcatttcc 180 ttttaactcc ttcctatcag gagtactttcttgtgtcgga actgctgttc ttgctgtttg 240 tctcaggatc caagtgaata aagagaataaggaattcaag gatcttgcac ctgagcgagc 300 ttttgcagat tttgttctct gtaatctggtgcttcatttg gtgatcatga acttccttgg 360 ttaatttgag ttcatgtggc tgttgttggttttgatcaaa ccttggataa taaaaagtaa 420 tagtagtata cctagacttt tgtaatagtatttatagaca gtaacttcca actaactgct 480 ttagtatttt gttgattcc 499 2 115 PRTPhaseolus lunatus 2 Met Ala Arg Ser Ser Ser Lys Asp Ala Gln Asp Leu PheArg Ala Leu 1 5 10 15 Trp Ser Ala Tyr Ala Ala Thr Pro Thr Asn Leu LysIle Ile Asp Leu 20 25 30 Tyr Val Met Phe Ala Val Phe Thr Ala Leu Ile GlnVal Val Tyr Met 35 40 45 Ala Leu Val Gly Ser Phe Pro Phe Asn Ser Phe LeuSer Gly Val Leu 50 55 60 Ser Cys Val Gly Thr Ala Val Leu Ala Val Cys LeuArg Ile Gln Val 65 70 75 80 Asn Lys Glu Asn Lys Glu Phe Lys Asp Leu AlaPro Glu Arg Ala Phe 85 90 95 Ala Asp Phe Val Leu Cys Asn Leu Val Leu HisLeu Val Ile Met Asn 100 105 110 Phe Leu Gly 115 3 712 DNA Zea mays 3gcacgagggc cgctgcccga ccccgacgcc tgctgcccag gtcttcgccg gcgacgagca 60ctccaccaga cgagagggga ttccaagatg ccgagggcca ccagcgacgc gaagctcctg 120atccagtccc tcggcaaggc gtacgctgcc acaccaacaa atctcaagat tattgacctc 180tacgtgggtt ttgcggttgc cactgccctt attcaggttg cttacatggg attggttggg 240tcgtttccct tcaactcctt cctctcagga gtcctttcat gcataggaac tgcagttctt 300gctgtttgcc tccgcattca agtgaacaaa gacaacaaag aattcaagga ccttccccca 360gaaagggcct ttgctgattt cgtcctatgc aatctggtgc tccacctggt gatcatgaat 420ttcctcggat aagcaactgc tgcaccatgt tggttaaagg ttttgtagcc ccaggttgtg 480gtcgctgatt gttgccttta aatgtttgga actgttgtga tcgtgatgtc gaatatccat 540atgatctgtt gaaggattac ttgtgtaagc tgagtattcc cggagggaac tattagtcga 600atggacagtt tgcccagcgc tgagaatgtg acctagcatg ttctttattt gaagaagata 660taattcattt ttcaaaaaaa aaaaaaaaaa aactcgaggg gggcccgtac cc 712 4 114 PRTZea mays 4 Met Pro Arg Ala Thr Ser Asp Ala Lys Leu Leu Ile Gln Ser LeuGly 1 5 10 15 Lys Ala Tyr Ala Ala Thr Pro Thr Asn Leu Lys Ile Ile AspLeu Tyr 20 25 30 Val Gly Phe Ala Val Ala Thr Ala Leu Ile Gln Val Ala TyrMet Gly 35 40 45 Leu Val Gly Ser Phe Pro Phe Asn Ser Phe Leu Ser Gly ValLeu Ser 50 55 60 Cys Ile Gly Thr Ala Val Leu Ala Val Cys Leu Arg Ile GlnVal Asn 65 70 75 80 Lys Asp Asn Lys Glu Phe Lys Asp Leu Pro Pro Glu ArgAla Phe Ala 85 90 95 Asp Phe Val Leu Cys Asn Leu Val Leu His Leu Val IleMet Asn Phe 100 105 110 Leu Gly 5 917 DNA Glycine max unsure (631) n isa, c, g or t 5 ggcaaatcgc gaagactaga tctgatctga gagaatggct cctcggtctagcagcaagga 60 cgcccaagac cttttccgcg ctctttggtc tgcttatgct gcaacccccactaatctcaa 120 gatcattgat ctctatgtca tctatgccgt attcaccgct ttcatccaggttgtttacat 180 ggctttggtt ggatcatttc catttaactc cttcctatca ggagtactttcttgtgtagg 240 aactgctgtt cttgctgttt gtctcaggat ccaagtgaat aaagagaataaggaattcaa 300 ggatcttgca cctgagcgcg cttttgcgga ttttgttctc tgtaatttggtgcttcattt 360 ggtgatcatg aacttccttg gttaaattgg gtttgtgtgg cggttgttgtttctgattga 420 acccttcgat aataaaaatt aaatagtagt atacctagac ttttgtaatagtatttatag 480 acagtagccg gcattctact aattgcttta gctttatgtt gattacccccacctcccatt 540 tttgggttcc ctgttttgaa cgaagagatt ttgccatctt ttgaagtttaaagtactttt 600 gaatggcgaa ataaagaagg attgttatta naaaanaaaa aaaaataacaanatatnaac 660 gcttacattt aagtggcact ttcggggaaa tgtgcgcgga accctattgttaatttccna 720 aatacatcaa atagtaccgc caagaacata accctgataa agctcataatatgaaaagga 780 gatanatatc acattcgtgt cgcctaaccc ttttgcggat ttgcctcccggttttgccaa 840 canaacccng gaaataaann gcgaaatact ggtgccaatg gtaacnatgnttcaaangga 900 aatctnaaat tcccaag 917 6 116 PRT Glycine max 6 Met AlaPro Arg Ser Ser Ser Lys Asp Ala Gln Asp Leu Phe Arg Ala 1 5 10 15 LeuTrp Ser Ala Tyr Ala Ala Thr Pro Thr Asn Leu Lys Ile Ile Asp 20 25 30 LeuTyr Val Ile Tyr Ala Val Phe Thr Ala Phe Ile Gln Val Val Tyr 35 40 45 MetAla Leu Val Gly Ser Phe Pro Phe Asn Ser Phe Leu Ser Gly Val 50 55 60 LeuSer Cys Val Gly Thr Ala Val Leu Ala Val Cys Leu Arg Ile Gln 65 70 75 80Val Asn Lys Glu Asn Lys Glu Phe Lys Asp Leu Ala Pro Glu Arg Ala 85 90 95Phe Ala Asp Phe Val Leu Cys Asn Leu Val Leu His Leu Val Ile Met 100 105110 Asn Phe Leu Gly 115 7 2671 DNA Zea mays unsure (690) n is a, c, g ort 7 gcatcccttc ccagttctgt ccctctcgaa ccctaactcc aaaaaccctc gctctcctct 60catggccgcc gccgcagctg acgacgcagc cgaggtggag cggctttacg agctcggcga 120gcgcctctcc tccgccaatg acaagtccga gcatgcggcg gactacgagg cgattattgc 180agcggtgaag ggacagagtg ccaaggcgaa gcagctcgca gcgcagctta tccccaggtt 240cttccggagc ttccctgcac tcggcacgcg cgccatgtca gccatgttcg atctcgtcga 300tatggaggag ctcgcgatca gaatacaagc tattcgtggc tttccacttc ttggcaaaga 360tactgaattt gtgtcaaaaa ttgcagatgt tttgggtcag ctccttacaa gcgaggaaaa 420tgttgagcgt gatgctgttc ataaagcgct catgtccctt atacggcaag atgttaaaaa 480ttcattacaa cctttattta agcatgtgga gcaaggatca gagattcgtg agaagattat 540ttgttttctt cgagacaagg tctttcctct taaagcagag ctgctgaaac ctcaagcaga 600aatggagaga tttataacgg atttgataaa gaaaagtgtg caagatgtaa ctggttcaga 660attcgaacta ttcatggggt tcttgcgaan ttggagcata tttggggatt ctgctcctag 720agagtccttt caagaactaa ttgaaattat tcaagcacag gctgatctga attcacaatt 780caacgtttct gacattgacc acattgagag gtggatttca tgcatgtata tggctcttcc 840gatcttcatg agaggagcat cagcaagcaa gttcctcaat tacttcgtta agcaaattgt 900tccagcattc gagaagattc ctgaagaaaa gaaactggat ttgctcaaga ctattgcttc 960aagttcaccg tacgcgacag ctcaagattc acgtcagctg cttccatctg ttgttcagtt 1020actcaacaaa tatatgcctg ggaagaaggt ggacgatatc aaccataatt atgttgaatg 1080cttgctgtac acttatcatc atttggctca taagactcca aacacaacga acagtctatg 1140tggttacaag attgttactg ggcaaccatc ggatagactt ggagaggact tcacagagca 1200ttacaaagat tttacagaga ggttaactgg aacagaagag acggtaagag cagcctcaaa 1260gcgactaact cagggaatgg cagatttcag caaggcaata tcttcagcaa aaaccgaaga 1320ggaaaaaact aaaattaaag gcgatcaaca aacttcaaca aggacaatga ggtcatataa 1380caatatattg gcaatgacac agtcattgca ttcaaaatcc cctttattta tcggtgataa 1440gaaaatcact ctgtcatgga tggagcagcc caacaaagca gcagctacga aagcaggggg 1500gaagaggtca caacctagta caaatgggaa tgaccctgca aacaagaagg ggagaggagg 1560aatgcaaaac cagctagtga acagagcttt tgaaggactg tctcatgttg gaagaggcag 1620tggaagaggt cggggcaagg gtgggccgag gaagaggaag aggatggagt ggggtaccac 1680tgagatgacc tggaaattat gttcaaacct gacaagggat gagctttctg cttccaccag 1740agagtaaact ccaaggtcga tgttttattg gtgtgcatta ctgcaacgcg ttgaaatgga 1800tcaaggcaca caacagcaga aatgcgtaca cagagaaagg atgctaagaa atatctgcaa 1860gtttgtgcat ctttcttatc catttaccat ctcatcgtgt tctttgccac cctaaccgtc 1920gtgtcacctg cgttggctgg ctgtttgatg aactgggcag ttcgatatct ttgttcttta 1980ttttatttta cagtgtttga agagacgacc aagcttgtgc tgactttgtt tgagttcgtt 2040ttatgtttcg tccttgtact gacacagatg tattagtgat gtttaacttt tatgtaacga 2100ttgattagct gtaatataag ttaagcattt aataagctat ttaaaaaaaa aaaaaaaacc 2160nkrcgggatt ggagttgggn cccactgagg atgaccttgg aaatttattg ttcaaaacct 2220tgaccaaggg gatgagcttt ctgcttccac cagagagtaa actccaaggt cgatgtttta 2280ttggtgtgca ttactgcaac gcgttgaaat ggatcaaggc acacaacagc agaaatgcgt 2340acacagagaa aggatgctaa gaaatatctg caagtttgtg catctttctt atccatttac 2400catctcatcg tgttctttgc caccctaacc gtcgtgtcac ctgcgttggc tggctgtttg 2460atgaactggg cagttcgata tctttgttct ttattttatt ttacagtgtt tgaagagacg 2520accaagcttg tgctgacttt gtttgagttc gttttatgtt tcgtccttgt actgacacag 2580atgtattagt gatgtttaac ttttatgtaa cgattgatta gctgtaatat aagttaagca 2640tttaataagc tatttaaaaa aaaaaaaaaa a 2671 8 561 PRT Zea mays UNSURE (210)Xaa can be any naturally occurring amino acid 8 Met Ala Ala Ala Ala AlaAsp Asp Ala Ala Glu Val Glu Arg Leu Tyr 1 5 10 15 Glu Leu Gly Glu ArgLeu Ser Ser Ala Asn Asp Lys Ser Glu His Ala 20 25 30 Ala Asp Tyr Glu AlaIle Ile Ala Ala Val Lys Gly Gln Ser Ala Lys 35 40 45 Ala Lys Gln Leu AlaAla Gln Leu Ile Pro Arg Phe Phe Arg Ser Phe 50 55 60 Pro Ala Leu Gly ThrArg Ala Met Ser Ala Met Phe Asp Leu Val Asp 65 70 75 80 Met Glu Glu LeuAla Ile Arg Ile Gln Ala Ile Arg Gly Phe Pro Leu 85 90 95 Leu Gly Lys AspThr Glu Phe Val Ser Lys Ile Ala Asp Val Leu Gly 100 105 110 Gln Leu LeuThr Ser Glu Glu Asn Val Glu Arg Asp Ala Val His Lys 115 120 125 Ala LeuMet Ser Leu Ile Arg Gln Asp Val Lys Asn Ser Leu Gln Pro 130 135 140 LeuPhe Lys His Val Glu Gln Gly Ser Glu Ile Arg Glu Lys Ile Ile 145 150 155160 Cys Phe Leu Arg Asp Lys Val Phe Pro Leu Lys Ala Glu Leu Leu Lys 165170 175 Pro Gln Ala Glu Met Glu Arg Phe Ile Thr Asp Leu Ile Lys Lys Ser180 185 190 Val Gln Asp Val Thr Gly Ser Glu Phe Glu Leu Phe Met Gly PheLeu 195 200 205 Arg Xaa Trp Ser Ile Phe Gly Asp Ser Ala Pro Arg Glu SerPhe Gln 210 215 220 Glu Leu Ile Glu Ile Ile Gln Ala Gln Ala Asp Leu AsnSer Gln Phe 225 230 235 240 Asn Val Ser Asp Ile Asp His Ile Glu Arg TrpIle Ser Cys Met Tyr 245 250 255 Met Ala Leu Pro Ile Phe Met Arg Gly AlaSer Ala Ser Lys Phe Leu 260 265 270 Asn Tyr Phe Val Lys Gln Ile Val ProAla Phe Glu Lys Ile Pro Glu 275 280 285 Glu Lys Lys Leu Asp Leu Leu LysThr Ile Ala Ser Ser Ser Pro Tyr 290 295 300 Ala Thr Ala Gln Asp Ser ArgGln Leu Leu Pro Ser Val Val Gln Leu 305 310 315 320 Leu Asn Lys Tyr MetPro Gly Lys Lys Val Asp Asp Ile Asn His Asn 325 330 335 Tyr Val Glu CysLeu Leu Tyr Thr Tyr His His Leu Ala His Lys Thr 340 345 350 Pro Asn ThrThr Asn Ser Leu Cys Gly Tyr Lys Ile Val Thr Gly Gln 355 360 365 Pro SerAsp Arg Leu Gly Glu Asp Phe Thr Glu His Tyr Lys Asp Phe 370 375 380 ThrGlu Arg Leu Thr Gly Thr Glu Glu Thr Val Arg Ala Ala Ser Lys 385 390 395400 Arg Leu Thr Gln Gly Met Ala Asp Phe Ser Lys Ala Ile Ser Ser Ala 405410 415 Lys Thr Glu Glu Glu Lys Thr Lys Ile Lys Gly Asp Gln Gln Thr Ser420 425 430 Thr Arg Thr Met Arg Ser Tyr Asn Asn Ile Leu Ala Met Thr GlnSer 435 440 445 Leu His Ser Lys Ser Pro Leu Phe Ile Gly Asp Lys Lys IleThr Leu 450 455 460 Ser Trp Met Glu Gln Pro Asn Lys Ala Ala Ala Thr LysAla Gly Gly 465 470 475 480 Lys Arg Ser Gln Pro Ser Thr Asn Gly Asn AspPro Ala Asn Lys Lys 485 490 495 Gly Arg Gly Gly Met Gln Asn Gln Leu ValAsn Arg Ala Phe Glu Gly 500 505 510 Leu Ser His Val Gly Arg Gly Ser GlyArg Xaa Arg Gly Xaa Gly Gly 515 520 525 Pro Arg Lys Arg Lys Arg Met GluTrp Gly Thr Thr Glu Met Thr Trp 530 535 540 Lys Leu Cys Ser Asn Leu ThrArg Asp Glu Leu Ser Ala Ser Thr Arg 545 550 555 560 Glu 9 556 DNA Oryzasativa unsure (179) n is a, c, g or t 9 ggcatactaa cccccccaaa tctcccacaccgctcccccg ccgccatggc cgcctccgac 60 gccgacgccg cggaggtcga gcggctctacgagctcggcg agcgcctctc ctccgccaag 120 gacaagtccc agcacgcggc ggactacgaggcgatcatat cggccgtgaa ggggcaganc 180 gtgaaggcga agcagctcgc ggcgcagctcatcccccgct tcttccggag cttcccggca 240 ctcgccccgc gcgccatgga ggccatgttcgacctcgtcg acatggatga actcgcgact 300 agaatacaac tattcgtggg ttttcacttcttgccaaana tgcanaattt gtctcaaaaa 360 ttgccganat ccttggacaa tccttgcaantnaggaaaat gtggacgtga tgctgtcata 420 aagcacngat gtcncttata cggcaggatttaaaattctt gcancttatt angattggat 480 tcgggatata attctnaaaa ttattgttcctaagaaangc tccngtaaag aaantgtgaa 540 ctcaacagag ngaaat 556 10 131 PRTOryza sativa UNSURE (45) Xaa can be any naturally occurring amino acid10 Met Ala Ala Ser Asp Ala Asp Ala Ala Glu Val Glu Arg Leu Tyr Glu 1 510 15 Leu Gly Glu Arg Leu Ser Ser Ala Lys Asp Lys Ser Gln His Ala Ala 2025 30 Asp Tyr Glu Ala Ile Ile Ser Ala Val Lys Gly Gln Xaa Val Lys Ala 3540 45 Lys Gln Leu Ala Ala Gln Leu Ile Pro Arg Phe Phe Arg Ser Phe Pro 5055 60 Ala Leu Ala Pro Arg Ala Met Glu Ala Met Phe Asp Leu Val Asp Met 6570 75 80 Asp Glu Leu Ala Thr Arg Ile Gln Leu Phe Val Gly Phe His Phe Leu85 90 95 Pro Xaa Met Xaa Asn Leu Ser Gln Lys Leu Pro Xaa Ser Leu Asp Asn100 105 110 Pro Cys Xaa Xaa Gly Lys Cys Gly Arg Asp Ala Val Ile Lys HisXaa 115 120 125 Cys Xaa Leu 130 11 749 DNA Glycine max unsure (678) n isa, c, g or t 11 gcgctggcta ctgtttatag tttactgtaa actgtgttta cgttgtgtcgtgtggcgtgt 60 tcagtgaggt aagggaaact cgtccccatc gaagagctta cttgacctcgcaccacggaa 120 tcgttcccta ctaattcaac tcaacaacac tatcgtctcc attcactagttagaaacgtg 180 cgttccaatg tctgatcctg ccgaagaggc tgctttcatc gagaagctctacgaatacgg 240 cgagcaactc aacaacacta tcgtctccat tcactagtta gaaacgtgcgttccaatgtc 300 tgatcctgcc gaagaggctg ctttcatcga gaagctctac gaatacggcgagcaactcaa 360 caatgccaag gacaagtcgc agaatgtgca ggattaccag ggaatcatagatgcggcgaa 420 gacgagtgtg aaggcgaagc agctcgctgc acagctgatt cccaggttctacaagttctt 480 tcctgacctt tctagccctg ctctcgatgc acatcttgat ttggttgaggctgaagaact 540 cggggttcga gtgcaagcaa ttagaggtct gcctcttttt tgtaaggatacacctgagaa 600 tattgggaag atggttgata ttcttgtgca aattcttggg tctgaggaatttgtggagcg 660 tgatgcagta cataaggntc ttaagtcctt tgctgaggca aggatgtcaaaagcttcctt 720 gacngctttg ttaagcacaa ttggnaagg 749 12 131 PRT Glycinemax UNSURE (128) Xaa can be any naturally occurring amino acid 12 MetSer Asp Pro Ala Glu Glu Ala Ala Phe Ile Glu Lys Leu Tyr Glu 1 5 10 15Tyr Gly Glu Gln Leu Asn Asn Ala Lys Asp Lys Ser Gln Asn Val Gln 20 25 30Asp Tyr Gln Gly Ile Ile Asp Ala Ala Lys Thr Ser Val Lys Ala Lys 35 40 45Gln Leu Ala Ala Gln Leu Ile Pro Arg Phe Tyr Lys Phe Phe Pro Asp 50 55 60Leu Ser Ser Pro Ala Leu Asp Ala His Leu Asp Leu Val Glu Ala Glu 65 70 7580 Glu Leu Gly Val Arg Val Gln Ala Ile Arg Gly Leu Pro Leu Phe Cys 85 9095 Lys Asp Thr Pro Glu Asn Ile Gly Lys Met Val Asp Ile Leu Val Gln 100105 110 Ile Leu Gly Ser Glu Glu Phe Val Glu Arg Asp Ala Val His Lys Xaa115 120 125 Leu Lys Ser 130 13 115 PRT Arabidopsis thaliana 13 Met ValLys Ser Thr Ser Lys Asp Ala Gln Asp Leu Phe His Ser Leu 1 5 10 15 HisSer Ala Tyr Thr Ala Thr Pro Thr Asn Leu Lys Ile Ile Asp Leu 20 25 30 TyrVal Cys Phe Ala Val Phe Thr Ala Leu Ile Gln Val Ala Tyr Met 35 40 45 AlaLeu Val Gly Ser Phe Pro Phe Asn Ser Phe Leu Ser Gly Val Leu 50 55 60 SerCys Ile Gly Thr Ala Val Leu Ala Val Cys Leu Arg Ile Gln Val 65 70 75 80Asn Lys Glu Asn Lys Glu Phe Lys Asp Leu Ala Pro Glu Arg Ala Phe 85 90 95Ala Asp Phe Val Leu Cys Asn Leu Val Leu His Leu Val Ile Ile Asn 100 105110 Phe Leu Gly 115 14 114 PRT Oryza sativa 14 Met Pro Arg Ala Thr SerAsp Ala Lys Leu Leu Ile Gln Ser Leu Gly 1 5 10 15 Lys Ala Tyr Ala AlaThr Pro Thr Asn Leu Lys Ile Ile Asp Leu Tyr 20 25 30 Val Val Phe Ala ValAla Thr Ala Leu Ile Gln Val Val Tyr Met Gly 35 40 45 Ile Val Gly Ser PhePro Phe Asn Ser Phe Leu Ser Gly Val Leu Ser 50 55 60 Cys Ile Gly Thr AlaVal Leu Ala Val Cys Leu Arg Ile Gln Val Asn 65 70 75 80 Lys Asp Asn LysGlu Phe Lys Asp Leu Pro Pro Glu Arg Ala Phe Ala 85 90 95 Asp Phe Val LeuCys Asn Leu Val Leu His Leu Val Ile Met Asn Phe 100 105 110 Leu Gly 15117 PRT Pisum sativum 15 Met Ala Lys Thr Ser Ser Thr Thr Lys Asp Ala GlnAsp Leu Phe His 1 5 10 15 Ala Ile Trp Ser Ala Tyr Ser Ala Thr Pro ThrAsn Leu Lys Ile Ile 20 25 30 Asp Leu Tyr Val Val Phe Ala Val Phe Thr AlaLeu Leu Gln Asp Val 35 40 45 Tyr Met Ala Leu Val Gly Pro Phe Pro Phe AsnSer Phe Leu Ser Gly 50 55 60 Val Leu Ser Cys Val Gly Thr Ala Val Leu AlaVal Cys Leu Arg Ile 65 70 75 80 Gln Val Asn Lys Glu Asn Lys Glu Phe LysAsp Leu Gly Pro Glu Arg 85 90 95 Ala Phe Ala Asp Phe Val Leu Cys Asn LeuVal Leu His Leu Val Ile 100 105 110 Met Asn Phe Leu Gly 115 16 528 PRTHomo sapiens 16 Met Gly Leu Ser Leu Thr Met Pro Thr Val Glu Glu Leu TyrArg Asn 1 5 10 15 Tyr Gly Ile Leu Ala Asp Ala Thr Glu Gln Val Gly GlnHis Lys Asp 20 25 30 Ala Tyr Gln Val Ile Leu Asp Gly Val Lys Gly Gly ThrLys Glu Lys 35 40 45 Arg Leu Ala Ala Gln Phe Ile Pro Lys Phe Phe Lys HisPhe Pro Glu 50 55 60 Leu Ala Asp Ser Ala Ile Asn Ala Gln Leu Asp Leu CysGlu Asp Glu 65 70 75 80 Asp Val Ser Ile Arg Arg Gln Ala Ile Lys Glu LeuPro Gln Phe Ala 85 90 95 Thr Gly Glu Asn Leu Pro Arg Val Ala Asp Ile LeuThr Gln Leu Leu 100 105 110 Gln Thr Asp Asp Ser Ala Glu Phe Asn Leu ValAsn Asn Ala Leu Leu 115 120 125 Ser Ile Phe Lys Met Asp Ala Lys Gly ThrLeu Gly Gly Leu Phe Ser 130 135 140 Gln Ile Leu Gln Gly Glu Asp Ile ValArg Glu Arg Ala Ile Lys Phe 145 150 155 160 Leu Ser Thr Lys Leu Lys ThrLeu Pro Asp Glu Val Leu Thr Lys Glu 165 170 175 Val Glu Glu Leu Ile LeuThr Glu Ser Lys Lys Val Leu Glu Asp Val 180 185 190 Thr Gly Glu Glu PheVal Leu Phe Met Lys Ile Leu Ser Gly Leu Lys 195 200 205 Ser Leu Gln ThrVal Ser Gly Arg Gln Gln Leu Val Glu Leu Val Ala 210 215 220 Glu Gln AlaAsp Leu Glu Gln Thr Phe Asn Pro Ser Asp Pro Asp Cys 225 230 235 240 ValAsp Arg Leu Leu Gln Cys Thr Arg Gln Ala Val Pro Leu Phe Ser 245 250 255Lys Asn Val His Ser Thr Arg Phe Val Thr Tyr Phe Cys Glu Gln Val 260 265270 Leu Pro Asn Leu Gly Thr Leu Thr Thr Pro Val Glu Gly Leu Asp Ile 275280 285 Gln Leu Glu Val Leu Lys Leu Leu Ala Glu Met Ser Ser Phe Cys Gly290 295 300 Asp Met Glu Lys Leu Glu Thr Asn Leu Arg Lys Leu Phe Asp LysLeu 305 310 315 320 Leu Glu Tyr Met Pro Leu Pro Pro Glu Glu Ala Glu AsnGly Glu Asn 325 330 335 Ala Gly Asn Glu Glu Pro Lys Leu Gln Phe Ser TyrVal Glu Cys Leu 340 345 350 Leu Tyr Ser Phe His Gln Leu Gly Arg Lys LeuPro Asp Phe Leu Thr 355 360 365 Ala Lys Leu Asn Ala Glu Lys Leu His GluSer Lys Ile Arg Leu Gln 370 375 380 Tyr Phe Ala Arg Gly Leu Gln Val TyrIle Arg Gln Leu Arg Leu Ala 385 390 395 400 Leu Gln Gly Lys Thr Gly GluAla Leu Lys Thr Glu Glu Asn Lys Ile 405 410 415 Lys Val Val Ala Leu LysIle Thr Asn Asn Ile Asn Val Leu Ile Lys 420 425 430 Asp Leu Phe His IlePro Pro Ser Tyr Lys Ser Thr Val Thr Leu Ser 435 440 445 Trp Lys Pro ValGln Lys Val Glu Ile Gly Gln Lys Arg Ala Ser Glu 450 455 460 Asp Thr ThrSer Gly Ser Pro Pro Lys Lys Ser Ser Ala Gly Pro Lys 465 470 475 480 ArgMet Pro Gly Arg Phe Ile Thr Leu Pro Val Gly Asn Ile Ala Ala 485 490 495Ile Trp Ala Thr Leu Ile Met Arg Gly Ala Phe Arg Gly Ser Lys Trp 500 505510 Pro Arg Leu Gly His Thr Lys Glu Ile Val Val Gly Glu Arg Leu Tyr 515520 525

1. A DNA sequence of SEQ ID NOS: 7, 8, 10, 11, 13, 14, 16, or 17encoding a fusion protein optionally having an N-terminal methionine,said fusion protein comprising an antibody constant region or portionthereof attached to the N-terminus of a human OB protein, wherein saidOB protein is selected from the group consisting of: (a) the amino acidsequence 1-146 as set forth in SEQ ID NO: 6; (b) the amino acid sequence1-146 as set forth in SEQ ID NO: 6 having a lysine residue at position35 and an isoleucine residue at position 74; (c) the amino acid sequenceof subparts (a) or (b) lacking a glutaminyl residue at position 28; and(d) the amino acid sequence of subparts (a), (b), or (c), wherein one ormore amino acids selected from the group consisting of amino acids 32,35, 50, 64, 68, 71, 74, 77, 89, 97, 100, 105, 106, 107, 108, 111, 118,136, 138, 142 and 145 is substituted with the corresponding amino acidpresent in SEQ ID NO: 3 or a conserved amino acid.
 2. The DNA sequenceencoding a fusion protein of claim 1, wherein the process of producingthe protein comprises expressing the DNA sequence in a transformed hostcell and isolating the fusion protein.
 3. A vector comprising the DNAsequence of claim
 1. 4. A host cell transformed or transfected with theDNA sequence of claim
 1. 5. The host cell of claim 4, wherein the hostcell produces a protein for use in treating obesity.
 6. The host cell ofclaim 5, wherein the protein is produced using the DNA sequence ofclaim
 1. 7. The host cell of claim 4, wherein the host cell produces aprotein for use in increasing insulin sensitivity.
 8. The host cell ofclaim 7, wherein the protein is produced using the DNA sequence of claim1.